Root-preferred promoter from a brachypodium distachyon metallothionein-like gene

ABSTRACT

This disclosure concerns compositions and methods for promoting transcription of a nucleotide sequence in a plant or plant cell, employing a promoter from a  Brachypodium distachyon  metallothionein-like gene (mtl). Some embodiments relate to a promoter from a  Brachypodium distachyon  metallothionein-like gene (mtl) that functions in plants to promote transcription of operably linked nucleotide sequences.

CROSS REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. patentapplication Ser. No. 15/097,345 filed Apr. 13, 2016, now allowed, whichclaims priority to U.S. Provisional Patent Application No. 62/147,868filed Apr. 15, 2015. The contents of the entirety of each of theforegoing are hereby incorporated in their entireties herein by thisreference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 36.7 KB ACII (Text) file named “77035-US-NP-20160322-Sequence-Listing-ST25.txt” created on Mar. 22, 2016.

FIELD OF THE DISCLOSURE

The present disclosure generally relates to compositions and methods forpromoting transcription of a nucleotide sequence in a plant or plantcell. Some embodiments relate to a promoter from a Brachypodiumdistachyon metallothionein-like gene (mtl) that functions in plants topromote transcription of an operably linked nucleotide sequence.Particular embodiments relate to methods including a promoter (e.g., tointroduce a nucleic acid molecule into a cell) and cells, cell cultures,tissues, organisms, and parts of organisms comprising a promoter, aswell as products produced therefrom.

BACKGROUND

Many plant species are capable of being transformed with transgenes tointroduce agronomically desirable traits or characteristics. Plantspecies are developed and/or modified to have particular desirabletraits. Generally, desirable traits include, for example, improvingnutritional value quality, increasing yield, conferring pest or diseaseresistance, increasing drought and stress tolerance, improvinghorticultural qualities (e.g., pigmentation and growth), impartingherbicide tolerance, enabling the production of industrially usefulcompounds and/or materials from the plant, and/or enabling theproduction of pharmaceuticals.

Transgenic plant species comprising multiple transgenes stacked at asingle genomic locus are produced via plant transformation technologies.Plant transformation technologies result in the introduction of atransgene into a plant cell, recovery of a fertile transgenic plant thatcontains the stably integrated copy of the transgene in the plantgenome, and subsequent transgene expression via transcription andtranslation of the plant genome results in transgenic plants thatpossess desirable traits and phenotypes. However, mechanisms that allowthe production of transgenic plant species to highly express multipletransgenes engineered as a trait stack are desirable.

Likewise, mechanisms that allow the expression of a transgene withinparticular tissues or organs of a plant are desirable. For example,increased resistance of a plant to infection by soil-borne pathogensmight be accomplished by transforming the plant genome with apathogen-resistance gene such that pathogen-resistance protein isrobustly expressed within the roots of the plant. Alternatively, it maybe desirable to express a transgene in plant tissues that are in aparticular growth or developmental phase such as, for example, celldivision or elongation. Furthermore, it may be desirable to express atransgene in leaf and stem tissues of a plant to provide toleranceagainst herbicides, or resistance against above ground insects andpests.

Therefore, a need exists for new gene regulatory elements that can drivethe desired levels of expression of transgenes in specific planttissues.

BRIEF SUMMARY

In embodiments of the subject disclosure, the disclosure relates to anucleic acid vector comprising a promoter operably linked to: apolylinker sequence; a non metallothionein-like gene; or a combinationof the polylinker sequence and the a non metallothionein-like gene,wherein said promoter comprises a polynucleotide sequence that has atleast 90% sequence identity with SEQ ID NO:1. In some embodiments, thepromoter is 2,000 bp in length. In additional embodiments, the promoterconsists of a polynucleotide sequence that has at least 90% sequenceidentity with SEQ ID NO:1. In other embodiments, the promoter drivesexpression of a polynucleotide encoding a selectable maker. In furtherembodiments, the promoter is operably linked to a transgene. In aspectsof this embodiment, the transgene encodes a selectable marker or a geneproduct conferring insecticidal resistance, herbicide tolerance,nitrogen use efficiency, water use efficiency, or nutritional quality.The promoter of SEQ ID NO:1 is provided for use with a 3′ untranslatedpolynucleotide sequence (3′-UTR), the 3′ untranslated polynucleotidesequence comprising a sequence that has at least 90% sequence identitywith SEQ ID NO:3, wherein the 3′ untranslated sequence is operablylinked to said polylinker or said transgene. In other embodiments, thepromoter of SEQ ID NO:1 is provided for use with a 5′ untranslatedpolynucleotide sequence, the 5′ untranslated polynucleotide sequencecomprising a sequence that has at least 90% sequence identity with SEQID NO:4, wherein the 5′ untranslated sequence is operably linked to saidpolylinker or said transgene. In other embodiments, the promoter of SEQID NO:1 further comprises an intron sequence. In a further embodiment,the promoter of SEQ ID NO:1 drives below ground tissue specificexpression.

In yet another embodiment, the subject disclosure provides for anon-Brachypodium plant comprising a polynucleotide sequence that has atleast 90% sequence identity with SEQ ID NO:1 operably linked to atransgene. In accordance with this embodiment, the plant is selectedfrom the group consisting of maize, wheat, rice, sorghum, oats, rye,bananas, sugar cane, soybean, cotton, Arabidopsis, tobacco, sunflower,and canola. Subsequently, the non-Brachypodium plant that comprises thepolynucleotide sequence that has at least 90% sequence identity with SEQID NO:1 may be a Zea mays plant in some embodiments. In otherembodiments, the transgene is that is operably linked to thepolynucleotide sequence that has at least 90% sequence identity with SEQID NO:1 is inserted into the genome of a plant. In some embodiments, thepolynucleotide sequence having at least 90% sequence identity with SEQID NO:1 is a promoter and said promoter is operably linked to atransgene. In other embodiments, the non-Brachypodium plant comprises a3′ untranslated sequence comprising SEQ ID NO:3 or a 3′ untranslatedsequence that has at least 90% sequence identity with SEQ ID NO:3,wherein the 3′ untranslated sequence is operably linked to saidtransgene. In an additional embodiment, the polynucleotide sequence thathas at least 90% sequence identity with SEQ ID NO:1 drives expression ofthe transgene with below ground tissue specific expression. In a furtherembodiment, the polynucleotide sequence that has at least 90% sequenceidentity with SEQ ID NO:1 is 2,000 bp in length.

In an embodiment, the subject disclosure provides for a method forproducing a transgenic plant cell, the method comprising the steps of:transforming a plant cell with a gene expression cassette comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoteroperably linked to at least one polynucleotide sequence of interest;isolating the transformed plant cell comprising the gene expressioncassette; and, producing a transgenic plant cell comprising theBrachypodium distachyon metallothionein-like gene (mtl) promoteroperably linked to at least one polynucleotide sequence of interest. Inother embodiments, the step of transforming a plant cell is performedwith a plant transformation method. The plant transformation method canbe selected from the group consisting of an Agrobacterium-mediatedtransformation method, a biolistics transformation method, a siliconcarbide transformation method, a protoplast transformation method, and aliposome transformation method. In other embodiments, the polynucleotidesequence of interest is constitutively expressed throughout thetransgenic plant cell. In some embodiments, the polynucleotide sequenceof interest is stably integrated into the genome of the transgenic plantcell. Accordingly, the method for producing a transgenic plant cell canfurther comprise the steps of: regenerating the transgenic plant cellinto a transgenic plant; and, obtaining the transgenic plant, whereinthe transgenic plant comprises the gene expression cassette comprisingthe Brachypodium distachyon metallothionein-like gene (mtl) promoteroperably linked to at least one polynucleotide sequence of interest. Inan embodiment, the transgenic plant cell is a monocotyledonoustransgenic plant cell or a dicotyledonous transgenic plant cell. Forexample, the dicotyledonous transgenic plant cell can be selected fromthe group consisting of an Arabidopsis plant cell, a tobacco plant cell,a soybean plant cell, a canola plant cell, and a cotton plant cell.Further, the monocotyledonous transgenic plant cell is selected from thegroup consisting of a maize plant cell, a rice plant cell, and a wheatplant cell. The Brachypodium distachyon metallothionein-like gene (mtl)promoter used in the method may comprise the polynucleotide of SEQ IDNO:1. In embodiments, the Brachypodium distachyon metallothionein-likegene (mtl) promoter may further comprise a first polynucleotide sequenceof interest operably linked to the 3′ end of SEQ ID NO:1.

In an embodiment, the subject disclosure provides for a method forexpressing a polynucleotide sequence of interest in a plant cell, themethod comprising introducing into the plant cell a polynucleotidesequence of interest operably linked to a Brachypodium distachyonmetallothionein-like gene (mtl) promoter. In some embodiments, thepolynucleotide sequence of interest operably linked to the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter is introduced intothe plant cell by a plant transformation method. As such, the planttransformation method can be selected from the group consisting of anAgrobacterium-mediated transformation method, a biolisticstransformation method, a silicon carbide transformation method, aprotoplast transformation method, and a liposome transformation method.In embodiments, the polynucleotide sequence of interest isconstitutively expressed throughout the plant cell. In some embodiments,the polynucleotide sequence of interest is stably integrated into thegenome of the plant cell. As such, the transgenic plant cell is amonocotyledonous plant cell or a dicotyledonous plant cell. As anexample, the dicotyledonous plant cell is selected from the groupconsisting of an Arabidopsis plant cell, a tobacco plant cell, a soybeanplant cell, a canola plant cell, and a cotton plant cell. Further, themonocotyledonous plant cell is selected from the group consisting of amaize plant cell, a rice plant cell, and a wheat plant cell.

In an embodiment, the subject disclosure provides for a transgenic plantcell comprising a Brachypodium distachyon metallothionein-like gene(mtl) promoter. In some embodiments, the transgenic plant cell comprisesa transgenic event. In an aspect of the embodiment, the transgenic eventcomprises an agronomic trait. Accordingly, the agronomic trait isselected from the group consisting of an insecticidal resistance trait,herbicide tolerance trait, nitrogen use efficiency trait, water useefficiency trait, nutritional quality trait, DNA binding trait,selectable marker trait, small RNA trait, or any combination thereof. Inother embodiments, the agronomic trait comprises an herbicide toleranttrait. In an aspect of the embodiment, the herbicide tolerant traitcomprises an aad-1 coding sequence. In some embodiments, the transgenicplant cell produces a commodity product. The commodity product isselected protein concentrate, protein isolate, grain, meal, flour, oil,or fiber. In an embodiment, the transgenic plant cell is selected fromthe group consisting of a dicotyledonous plant cell or amonocotyledonous plant cell. Accordingly, the monocotyledonous plantcell is a maize plant cell. In other embodiments, the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter comprises apolynucleotide with at least 90% sequence identity to the polynucleotideof SEQ ID NO:1. In yet another embodiment, the Brachypodium distachyonmetallothionein-like gene (mtl) promoter is 2,000 bp in length. Infurther embodiments, the Brachypodium distachyon metallothionein-likegene (mtl) promoter consists of SEQ ID NO:1. In additional embodiments,the Brachypodium distachyon metallothionein-like gene (mtl) promoterconsists of SEQ ID NO:1 is operably linked to the 3′ end of SEQ ID NO:1.In other embodiments the Brachypodium distachyon metallothionein-likegene (mtl) promoter drives expression of an agronomic trait in belowground plant tissues.

The subject disclosure provides for an isolated polynucleotidecomprising a nucleic acid sequence with at least 90% sequence identityto the polynucleotide of SEQ ID NO:1. In some embodiments, the isolatedpolynucleotide drives below ground tissue specific expression. In otherembodiments, the isolated polynucleotide has expression activity withina plant cell. In embodiments, the isolated polynucleotide comprises anopen-reading frame polynucleotide coding for a polypeptide; and atermination sequence. Further embodiments include the isolatedpolynucleotide comprising a nucleic acid sequence with at least 90%sequence identity to the polynucleotide of SEQ ID NO:1, wherein thepolynucleotide of SEQ ID NO:1 is 2,000 bp in length.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: This figure is a schematic of pDAB102419 which contains theBrachypodium distachyon metallothionein-like gene (mtl) promoter of SEQID NO:1 (labeled as B distachyon MTL Promoter) and the Brachypodiumdistachyon metallothionein-like gene (mtl) 3′-UTR of SEQ ID NO:3(labeled as B distachyon MTL 3′UTR).

FIG. 2: This figure provides a polynucleotide alignment of theBrachypodium distachyon metallothionein-like gene (mtl) promoter of SEQID NO:1 (2,000 bp) and the truncated Brachypodium distachyonmetallothionein-like gene (mtl) promoters of SEQ ID NO:7 (1,730 bp), SEQID NO:8 (1,530 bp), SEQ ID NO:9 (1,330 bp), SEQ ID NO:10 (1,130 bp) andSEQ ID NO:11 (1,001 bp).

FIG. 3: This figure provides a polynucleotide alignment of theBrachypodium distachyon metallothionein-like gene (mtl) promoter of SEQID NO:1 and the modified Brachypodium distachyon metallothionein-likegene (mtl) promoter of SEQ ID NO:12.

FIG. 4: This figure provides a polynucleotide alignment of theBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR of SEQ IDNO:3 and the truncated Brachypodium distachyon metallothionein-like gene(mtl) 3′-UTRs of SEQ ID NO:13 (264 bp), SEQ ID NO:14 (332 bp), SEQ IDNO:15 (630 bp), and SEQ ID NO:16 (727 bp).

FIG. 5: This figure provides a polynucleotide alignment of theBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR of SEQ IDNO:3 and the modified Brachypodium distachyon metallothionein-like gene(mtl) 3′-UTR of SEQ ID NO:17 and SEQ ID NO:18.

DETAILED DESCRIPTION

I. Overview of Several Embodiments

Development of transgenic plant products is becoming increasinglycomplex. Commercially viable transgenic plants now require the stackingof multiple transgenes into a single locus. Plant promoters used forbasic research or biotechnological applications are generallyunidirectional, directing only one gene that has been fused at its 3′end (downstream). Accordingly, each transgene usually requires apromoter for expression, wherein multiple promoters are required toexpress multiple transgenes within one gene stack. With an increasingnumber of transgenes in gene stacks, the same promoter is routinely usedto obtain similar levels of expression patterns of different transgenes.Obtaining similar levels of transgene expression is necessary for theproduction of a single polygenic trait. Unfortunately, multi-geneconstructs driven by the same promoter are known to cause gene silencingresulting in less efficacious transgenic products in the field. Therepeated promoter elements may lead to homology-based gene silencing. Inaddition, repetitive sequences within a transgene may lead to gene intralocus homologous recombination resulting in polynucleotiderearrangements. The silencing and rearrangement of transgenes willlikely have an undesirable affect on the performance of a transgenicplant produced to express transgenes. Further, excess of transcriptionfactor (TF)-binding sites due to promoter repetition can cause depletionof endogenous TFs leading to transcriptional inactivation. Given theneed to introduce multiple genes into plants for metabolic engineeringand trait stacking, a variety of promoters are required to developtransgenic crops that drive the expression of multiple genes.

A particular problem in promoter identification is the need to identifytissue-specific promoters, related to specific cell types, developmentalstages and/or functions in the plant that are not expressed in otherplant tissues. Tissue specific (i.e., tissue preferred) or organspecific promoters drive gene expression in a certain tissue such as inthe kernel, root, leaf, or tapetum of the plant. Tissue anddevelopmental stage specific promoters can be initially identified fromobserving the expression of genes, which are expressed in particulartissues or at particular time periods during plant development. Thesetissue specific promoters are required for certain applications in thetransgenic plant industry and are desirable as they permit specificexpression of heterologous genes in a tissue and/or developmental stageselective manner, indicating expression of the heterologous genedifferentially at various organs, tissues and/or times, but not in othertissue. For example, increased resistance of a plant to infection bysoil-borne pathogens might be accomplished by transforming the plantgenome with a pathogen-resistance gene such that pathogen-resistanceprotein is robustly expressed within the roots of the plant.Alternatively, it may be desirable to express a transgene in planttissues that are in a particular growth or developmental phase such as,for example, cell division or elongation. Another application is thedesirability of using tissue specific promoters to confine theexpression of the transgenes encoding an agronomic trait in specifictissues types like developing parenchyma cells. As such, a particularproblem in the identification of promoters is how to identify thepromoters, and to relate the identified promoter to developmentalproperties of the cell for specific tissue expression.

Another problem regarding the identification of a promoter is therequirement to clone all relevant cis-acting and trans-activatingtranscriptional control elements so that the cloned DNA fragment drivestranscription in the wanted specific expression pattern. Given that suchcontrol elements are located distally from the translation initiation orstart site, the size of the polynucleotide that is selected to comprisethe promoter is of importance for providing the level of expression andthe expression patterns of the promoter polynucleotide sequence. It isknown that promoter lengths include functional information, anddifferent genes have been shown to have promoters longer or shorter thanpromoters of the other genes in the genome. Elucidating thetranscription start site of a promoter and predicting the functionalgene elements in the promoter region is challenging. Further adding tothe challenge are the complexity, diversity and inherent degeneratenature of regulatory motifs and cis- and trans-regulatory elements(Blanchette, Mathieu, et al. “Genome-wide computational prediction oftranscriptional regulatory modules reveals new insights into human geneexpression.” Genome research 16.5 (2006): 656-668). The cis- andtrans-regulatory elements are located in the distal parts of thepromoter which regulate the spatial and temporal expression of a gene tooccur only at required sites and at specific times (Porto, Milena Silva,et al. “Plant promoters: an approach of structure and function.”Molecular biotechnology 56.1 (2014): 38-49). Existing promoter analysistools cannot reliably identify such cis regulatory elements in a genomicsequence, thus predicting too many false positives because these toolsare generally focused only on the sequence content (Fickett J W,Hatzigeorgiou A G (1997) Eukaryotic promoter recognition. Genomeresearch 7: 861-878). Accordingly, the identification of promoterregulatory elements requires that an appropriate sequence of a specificsize is obtained that will result in driving expression of an operablylinked transgene in a desirable manner.

Provided are methods and compositions for overcoming such problemsthrough the use of Brachypodium distachyon metallothionein-like gene(mtl) promoter regulatory elements to express transgenes in plant.

II. Terms and Abbreviations

Throughout the application, a number of terms are used. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

As used herein, the term “intron” refers to any nucleic acid sequencecomprised in a gene (or expressed polynucleotide sequence of interest)that is transcribed but not translated. Introns include untranslatednucleic acid sequence within an expressed sequence of DNA, as well asthe corresponding sequence in RNA molecules transcribed therefrom. Aconstruct described herein can also contain sequences that enhancetranslation and/or mRNA stability such as introns. An example of onesuch intron is the first intron of gene II of the histone H3 variant ofArabidopsis thaliana or any other commonly known intron sequence.Introns can be used in combination with a promoter sequence to enhancetranslation and/or mRNA stability.

The term “isolated”, as used herein means having been removed from itsnatural environment, or removed from other compounds present when thecompound is first formed. The term “isolated” embraces materialsisolated from natural sources as well as materials (e.g., nucleic acidsand proteins) recovered after preparation by recombinant expression in ahost cell, or chemically-synthesized compounds such as nucleic acidmolecules, proteins, and peptides.

The term “purified”, as used herein relates to the isolation of amolecule or compound in a form that is substantially free ofcontaminants normally associated with the molecule or compound in anative or natural environment, or substantially enriched inconcentration relative to other compounds present when the compound isfirst formed, and means having been increased in purity as a result ofbeing separated from other components of the original composition. Theterm “purified nucleic acid” is used herein to describe a nucleic acidsequence which has been separated, produced apart from, or purified awayfrom other biological compounds including, but not limited topolypeptides, lipids and carbohydrates, while effecting a chemical orfunctional change in the component (e.g., a nucleic acid may be purifiedfrom a chromosome by removing protein contaminants and breaking chemicalbonds connecting the nucleic acid to the remaining DNA in thechromosome).

The term “synthetic”, as used herein refers to a polynucleotide (i.e., aDNA or RNA) molecule that was created via chemical synthesis as an invitro process. For example, a synthetic DNA may be created during areaction within an Eppendorf™ tube, such that the synthetic DNA isenzymatically produced from a native strand of DNA or RNA. Otherlaboratory methods may be utilized to synthesize a polynucleotidesequence. Oligonucleotides may be chemically synthesized on an oligosynthesizer via solid-phase synthesis using phosphoramidites. Thesynthesized oligonucleotides may be annealed to one another as acomplex, thereby producing a “synthetic” polynucleotide. Other methodsfor chemically synthesizing a polynucleotide are known in the art, andcan be readily implemented for use in the present disclosure.

The term “about” as used herein means greater or lesser than the valueor range of values stated by 10 percent, but is not intended todesignate any value or range of values to only this broader definition.Each value or range of values preceded by the term “about” is alsointended to encompass the embodiment of the stated absolute value orrange of values.

For the purposes of the present disclosure, a “gene,” includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

As used herein the terms “native” or “natural” define a condition foundin nature. A “native DNA sequence” is a DNA sequence present in naturethat was produced by natural means or traditional breeding techniquesbut not generated by genetic engineering (e.g., using molecularbiology/transformation techniques).

As used herein a “transgene” is defined to be a nucleic acid sequencethat encodes a gene product, including for example, but not limited to,an mRNA. In one embodiment the transgene is an exogenous nucleic acid,where the transgene sequence has been introduced into a host cell bygenetic engineering (or the progeny thereof) where the transgene is notnormally found. In one example, a transgene encodes an industrially orpharmaceutically useful compound, or a gene encoding a desirableagricultural trait (e.g., an herbicide-resistance gene). In yet anotherexample, a transgene is an antisense nucleic acid sequence, whereinexpression of the antisense nucleic acid sequence inhibits expression ofa target nucleic acid sequence. In one embodiment the transgene is anendogenous nucleic acid, wherein additional genomic copies of theendogenous nucleic acid are desired, or a nucleic acid that is in theantisense orientation with respect to the sequence of a target nucleicacid in a host organism.

As used herein the term “non metallothionein-like transgene” or “nonmetallothionein-like gene” is any transgene that has less than 80%sequence identity with the Brachypodium distachyon metallothionein-likegene coding sequence (SEQ ID NO:6).

A “gene product” as defined herein is any product produced by the gene.For example the gene product can be the direct transcriptional productof a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, interfering RNA,ribozyme, structural RNA or any other type of RNA) or a protein producedby translation of a mRNA. Gene products also include RNAs which aremodified, by processes such as capping, polyadenylation, methylation,and editing, and proteins modified by, for example, methylation,acetylation, phosphorylation, ubiquitination, ADP-ribosylation,myristilation, and glycosylation. Gene expression can be influenced byexternal signals, for example, exposure of a cell, tissue, or organismto an agent that increases or decreases gene expression. Expression of agene can also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

As used herein the term “gene expression” relates to the process bywhich the coded information of a nucleic acid transcriptional unit(including, e.g., genomic DNA) is converted into an operational,non-operational, or structural part of a cell, often including thesynthesis of a protein. Gene expression can be influenced by externalsignals; for example, exposure of a cell, tissue, or organism to anagent that increases or decreases gene expression. Expression of a genecan also be regulated anywhere in the pathway from DNA to RNA toprotein. Regulation of gene expression occurs, for example, throughcontrols acting on transcription, translation, RNA transport andprocessing, degradation of intermediary molecules such as mRNA, orthrough activation, inactivation, compartmentalization, or degradationof specific protein molecules after they have been made, or bycombinations thereof. Gene expression can be measured at the RNA levelor the protein level by any method known in the art, including, withoutlimitation, Northern blot, RT-PCR, Western blot, or in vitro, in situ,or in vivo protein activity assay(s).

As used herein, “homology-based gene silencing” (HBGS) is a generic termthat includes both transcriptional gene silencing andpost-transcriptional gene silencing. Silencing of a target locus by anunlinked silencing locus can result from transcription inhibition(transcriptional gene silencing; TGS) or mRNA degradation(post-transcriptional gene silencing; PTGS), owing to the production ofdouble-stranded RNA (dsRNA) corresponding to promoter or transcribedsequences, respectively. The involvement of distinct cellular componentsin each process suggests that dsRNA-induced TGS and PTGS likely resultfrom the diversification of an ancient common mechanism. However, astrict comparison of TGS and PTGS has been difficult to achieve becauseit generally relies on the analysis of distinct silencing loci. In someinstances, a single transgene locus can triggers both TGS and PTGS,owing to the production of dsRNA corresponding to promoter andtranscribed sequences of different target genes. Mourrain et al. (2007)Planta 225:365-79. It is likely that siRNAs are the actual moleculesthat trigger TGS and PTGS on homologous sequences: the siRNAs would inthis model trigger silencing and methylation of homologous sequences incis and in trans through the spreading of methylation of transgenesequences into the endogenous promoter.

As used herein, the term “nucleic acid molecule” (or “nucleic acid” or“polynucleotide”) may refer to a polymeric form of nucleotides, whichmay include both sense and anti-sense strands of RNA, cDNA, genomic DNA,and synthetic forms and mixed polymers of the above. A nucleotide mayrefer to a ribonucleotide, deoxyribonucleotide, or a modified form ofeither type of nucleotide. A “nucleic acid molecule” as used herein issynonymous with “nucleic acid” and “polynucleotide”. A nucleic acidmolecule is usually at least 10 bases in length, unless otherwisespecified. The term may refer to a molecule of RNA or DNA ofindeterminate length. The term includes single- and double-strandedforms of DNA. A nucleic acid molecule may include either or bothnaturally-occurring and modified nucleotides linked together bynaturally occurring and/or non-naturally occurring nucleotide linkages.

Nucleic acid molecules may be modified chemically or biochemically, ormay contain non-natural or derivatized nucleotide bases, as will bereadily appreciated by those of skill in the art. Such modificationsinclude, for example, labels, methylation, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications (e.g., uncharged linkages: for example, methylphosphonates, phosphotriesters, phosphoramidites, carbamates, etc.;charged linkages: for example, phosphorothioates, phosphorodithioates,etc.; pendent moieties: for example, peptides; intercalators: forexample, acridine, psoralen, etc.; chelators; alkylators; and modifiedlinkages: for example, alpha anomeric nucleic acids, etc.). The term“nucleic acid molecule” also includes any topological conformation,including single-stranded, double-stranded, partially duplexed,triplexed, hairpinned, circular, and padlocked conformations.

Transcription proceeds in a 5′ to 3′ manner along a DNA strand. Thismeans that RNA is made by the sequential addition ofribonucleotide-5′-triphosphates to the 3′ terminus of the growing chain(with a requisite elimination of the pyrophosphate). In either a linearor circular nucleic acid molecule, discrete elements (e.g., particularnucleotide sequences) may be referred to as being “upstream” or “5′”relative to a further element if they are bonded or would be bonded tothe same nucleic acid in the 5′ direction from that element. Similarly,discrete elements may be “downstream” or “3′” relative to a furtherelement if they are or would be bonded to the same nucleic acid in the3′ direction from that element.

A base “position”, as used herein, refers to the location of a givenbase or nucleotide residue within a designated nucleic acid. Thedesignated nucleic acid may be defined by alignment (see below) with areference nucleic acid.

Hybridization relates to the binding of two polynucleotide strands viaHydrogen bonds. Oligonucleotides and their analogs hybridize by hydrogenbonding, which includes Watson-Crick, Hoogsteen or reversed Hoogsteenhydrogen bonding, between complementary bases. Generally, nucleic acidmolecules consist of nitrogenous bases that are either pyrimidines(cytosine (C), uracil (U), and thymine (T)) or purines (adenine (A) andguanine (G)). These nitrogenous bases form hydrogen bonds between apyrimidine and a purine, and the bonding of the pyrimidine to the purineis referred to as “base pairing.” More specifically, A will hydrogenbond to T or U, and G will bond to C. “Complementary” refers to the basepairing that occurs between two distinct nucleic acid sequences or twodistinct regions of the same nucleic acid sequence.

“Specifically hybridizable” and “specifically complementary” are termsthat indicate a sufficient degree of complementarity such that stableand specific binding occurs between the oligonucleotide and the DNA orRNA target. The oligonucleotide need not be 100% complementary to itstarget sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget DNA or RNA molecule interferes with the normal function of thetarget DNA or RNA, and there is sufficient degree of complementarity toavoid non-specific binding of the oligonucleotide to non-targetsequences under conditions where specific binding is desired, forexample under physiological conditions in the case of in vivo assays orsystems. Such binding is referred to as specific hybridization.

Hybridization conditions resulting in particular degrees of stringencywill vary depending upon the nature of the chosen hybridization methodand the composition and length of the hybridizing nucleic acidsequences. Generally, the temperature of hybridization and the ionicstrength (especially the Na+ and/or Mg2+ concentration) of thehybridization buffer will contribute to the stringency of hybridization,though wash times also influence stringency. Calculations regardinghybridization conditions required for attaining particular degrees ofstringency are discussed in Sambrook et al. (ed.), Molecular Cloning: ALaboratory Manual, 2nd ed., vol. 1-3, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989, chs. 9 and 11.

As used herein, “stringent conditions” encompass conditions under whichhybridization will only occur if there is less than 50% mismatch betweenthe hybridization molecule and the DNA target. “Stringent conditions”include further particular levels of stringency. Thus, as used herein,“moderate stringency” conditions are those under which molecules withmore than 50% sequence mismatch will not hybridize; conditions of “highstringency” are those under which sequences with more than 20% mismatchwill not hybridize; and conditions of “very high stringency” are thoseunder which sequences with more than 10% mismatch will not hybridize.

In particular embodiments, stringent conditions can includehybridization at 65° C., followed by washes at 65° C. with 0.1×SSC/0.1%SDS for 40 minutes.

The following are representative, non-limiting hybridization conditions:

-   -   Very High Stringency: Hybridization in 5×SSC buffer at 65° C.        for 16 hours; wash twice in 2×SSC buffer at room temperature for        15 minutes each; and wash twice in 0.5×SSC buffer at 65° C. for        20 minutes each.    -   High Stringency: Hybridization in 5×-6×SSC buffer at 65-70° C.        for 16-20 hours; wash twice in 2×SSC buffer at room temperature        for 5-20 minutes each; and wash twice in 1×SSC buffer at        55-70° C. for 30 minutes each.    -   Moderate Stringency: Hybridization in 6×SSC buffer at room        temperature to 55° C. for 16-20 hours; wash at least twice in        2×-3×SSC buffer at room temperature to 55° C. for 20-30 minutes        each.

In particular embodiments, specifically hybridizable nucleic acidmolecules can remain bound under very high stringency hybridizationconditions. In these and further embodiments, specifically hybridizablenucleic acid molecules can remain bound under high stringencyhybridization conditions. In these and further embodiments, specificallyhybridizable nucleic acid molecules can remain bound under moderatestringency hybridization conditions.

Oligonucleotide: An oligonucleotide is a short nucleic acid polymer.Oligonucleotides may be formed by cleavage of longer nucleic acidsegments, or by polymerizing individual nucleotide precursors. Automatedsynthesizers allow the synthesis of oligonucleotides up to severalhundred base pairs in length. Because oligonucleotides may bind to acomplementary nucleotide sequence, they may be used as probes fordetecting DNA or RNA. Oligonucleotides composed of DNA(oligodeoxyribonucleotides) may be used in PCR, a technique for theamplification of small DNA sequences. In PCR, the oligonucleotide istypically referred to as a “primer”, which allows a DNA polymerase toextend the oligonucleotide and replicate the complementary strand.

As used herein, the term “sequence identity” or “identity”, as usedherein in the context of two nucleic acid or polypeptide sequences, mayrefer to the residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window.

As used herein, the term “percentage of sequence identity” may refer tothe value determined by comparing two optimally aligned sequences (e.g.,nucleic acid sequences, and amino acid sequences) over a comparisonwindow, wherein the portion of the sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleotide oramino acid residue occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the comparison window, and multiplying the resultby 100 to yield the percentage of sequence identity.

Methods for aligning sequences for comparison are well-known in the art.Various programs and alignment algorithms are described in, for example:Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch(1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad.Sci. U.S.A. 85:2444; Higgins and Sharp (1988) Gene 73:237-44; Higginsand Sharp (1989) CABIOS 5:151-3; Corpet et al. (1988) Nucleic Acids Res.16:10881-90; Huang et al. (1992) Comp. Appl. Biosci. 8:155-65; Pearsonet al. (1994) Methods Mol. Biol. 24:307-31; Tatiana et al. (1999) FEMSMicrobiol. Lett. 174:247-50. A detailed consideration of sequencealignment methods and homology calculations can be found in, e.g.,Altschul et al. (1990) J. Mol. Biol. 215:403-10.

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™; Altschul et al. (1990)) is available fromseveral sources, including the National Center for BiotechnologyInformation (Bethesda, Md.), and on the internet, for use in connectionwith several sequence analysis programs. A description of how todetermine sequence identity using this program is available on theinternet under the “help” section for BLAST™. For comparisons of nucleicacid sequences, the “Blast 2 sequences” function of the BLAST™ (Blastn)program may be employed using the default parameters. Nucleic acidsequences with even greater similarity to the reference sequences willshow increasing percentage identity when assessed by this method.

As used herein the term “operably linked” relates to a first nucleicacid sequence is operably linked with a second nucleic acid sequencewhen the first nucleic acid sequence is in a functional relationshipwith the second nucleic acid sequence. For instance, a promoter isoperably linked with a coding sequence when the promoter affects thetranscription or expression of the coding sequence. When recombinantlyproduced, operably linked nucleic acid sequences are generallycontiguous and, where necessary to join two protein-coding regions, inthe same reading frame. However, elements need not be contiguous to beoperably linked.

As used herein, the term “promoter” refers to a region of DNA thatgenerally is located upstream (towards the 5′ region of a gene) of agene and is needed to initiate and drive transcription of the gene. Apromoter may permit proper activation or repression of a gene that itcontrols. A promoter may contain specific sequences that are recognizedby transcription factors. These factors may bind to a promoter DNAsequence, which results in the recruitment of RNA polymerase, an enzymethat synthesizes RNA from the coding region of the gene. The promotergenerally refers to all gene regulatory elements located upstream of thegene, including, upstream promoters, 5′-UTR, introns, and leadersequences.

As used herein, the term “upstream-promoter” refers to a contiguouspolynucleotide sequence that is sufficient to direct initiation oftranscription. As used herein, an upstream-promoter encompasses the siteof initiation of transcription with several sequence motifs, whichinclude TATA Box, initiator sequence, TFIIB recognition elements andother promoter motifs (Jennifer, E. F. et al., (2002) Genes & Dev., 16:2583-2592). The upstream promoter provides the site of action to RNApolymerase II which is a multi-subunit enzyme with the basal or generaltranscription factors like, TFIIA, B, D, E, F and H. These factorsassemble into a transcription pre initiation complex that catalyzes thesynthesis of RNA from DNA template.

The activation of the upstream-promoter is done by the additionalsequence of regulatory DNA sequence elements to which various proteinsbind and subsequently interact with the transcription initiation complexto activate gene expression. These gene regulatory elements sequencesinteract with specific DNA-binding factors. These sequence motifs maysometimes be referred to as cis-elements. Such cis-elements, to whichtissue-specific or development-specific transcription factors bind,individually or in combination, may determine the spatiotemporalexpression pattern of a promoter at the transcriptional level. Thesecis-elements vary widely in the type of control they exert on operablylinked genes. Some elements act to increase the transcription ofoperably-linked genes in response to environmental responses (e.g.,temperature, moisture, and wounding). Other cis-elements may respond todevelopmental cues (e.g., germination, seed maturation, and flowering)or to spatial information (e.g., tissue specificity). See, for example,Langridge et al., (1989) Proc. Natl. Acad. Sci. USA 86:3219-23. Thesecis-elements are located at a varying distance from transcription startpoint, some cis-elements (called proximal elements) are adjacent to aminimal core promoter region while other elements can be positionedseveral kilobases upstream or downstream of the promoter (enhancers).

As used herein, the terms “5′ untranslated region” or “5′-UTR” isdefined as the untranslated segment in the 5′ terminus of pre-mRNAs ormature mRNAs. For example, on mature mRNAs, a 5′-UTR typically harborson its 5′ end a 7-methylguanosine cap and is involved in many processessuch as splicing, polyadenylation, mRNA export towards the cytoplasm,identification of the 5′ end of the mRNA by the translational machinery,and protection of the mRNAs against degradation.

As used herein, the terms “transcription terminator” is defined as thetranscribed segment in the 3′ terminus of pre-mRNAs or mature mRNAs. Forexample, longer stretches of DNA beyond “polyadenylation signal” site istranscribed as a pre-mRNA. This DNA sequence usually containstranscription termination signal for the proper processing of thepre-mRNA into mature mRNA.

As used herein, the term “3′ untranslated region” or “3′-UTR” is definedas the untranslated segment in a 3′ terminus of the pre-mRNAs or maturemRNAs. For example, on mature mRNAs this region harbors the poly-(A)tail and is known to have many roles in mRNA stability, translationinitiation, and mRNA export. In addition, the 3′-UTR is considered toinclude the polyadenylation signal and transcription terminator.

As used herein, the term “polyadenylation signal” designates a nucleicacid sequence present in mRNA transcripts that allows for transcripts,when in the presence of a poly-(A) polymerase, to be polyadenylated onthe polyadenylation site, for example, located 10 to 30 bases downstreamof the poly-(A) signal. Many polyadenylation signals are known in theart and are useful for the present invention. An exemplary sequenceincludes AAUAAA and variants thereof, as described in Loke J., et al.,(2005) Plant Physiology 138(3); 1457-1468.

A “DNA binding transgene” is a polynucleotide coding sequence thatencodes a DNA binding protein. The DNA binding protein is subsequentlyable to bind to another molecule. A binding protein can bind to, forexample, a DNA molecule (a DNA-binding protein), a RNA molecule (anRNA-binding protein), and/or a protein molecule (a protein-bindingprotein). In the case of a protein-binding protein, it can bind toitself (to form homodimers, homotrimers, etc.) and/or it can bind to oneor more molecules of a different protein or proteins. A binding proteincan have more than one type of binding activity. For example, zincfinger proteins have DNA-binding, RNA-binding, and protein-bindingactivity.

Examples of DNA binding proteins include; meganucleases, zinc fingers,CRISPRs, and TALE binding domains that can be “engineered” to bind to apredetermined nucleotide sequence. Typically, the engineered DNA bindingproteins (e.g., zinc fingers, CRISPRs, or TALEs) are proteins that arenon-naturally occurring. Non-limiting examples of methods forengineering DNA-binding proteins are design and selection. A designedDNA binding protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP, CRISPR, and/or TALE designs and bindingdata. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536and WO 03/016496 and U.S. Publication Nos. 20110301073, 20110239315 and20119145940.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP. Zinc finger bindingdomains can be “engineered” to bind to a predetermined nucleotidesequence. Non-limiting examples of methods for engineering zinc fingerproteins are design and selection. A designed zinc finger protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPdesigns and binding data. See, for example, U.S. Pat. Nos. 6,140,081;6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496.

In other examples, the DNA-binding domain of one or more of thenucleases comprises a naturally occurring or engineered (non-naturallyoccurring) TAL effector DNA binding domain. See, e.g., U.S. PatentPublication No. 20110301073, incorporated by reference in its entiretyherein. The plant pathogenic bacteria of the genus Xanthomonas are knownto cause many diseases in important crop plants. Pathogenicity ofXanthomonas depends on a conserved type III secretion (T3S) system whichinjects more than different effector proteins into the plant cell. Amongthese injected proteins are transcription activator-like (TALEN)effectors which mimic plant transcriptional activators and manipulatethe plant transcriptome (see Kay et al., (2007) Science 318:648-651).These proteins contain a DNA binding domain and a transcriptionalactivation domain. One of the most well characterized TAL-effectors isAvrBs3 from Xanthomonas campestgris pv. Vesicatoria (see Bonas et al.,(1989) Mol Gen Genet 218: 127-136 and WO2010079430). TAL-effectorscontain a centralized domain of tandem repeats, each repeat containingapproximately 34 amino acids, which are key to the DNA bindingspecificity of these proteins. In addition, they contain a nuclearlocalization sequence and an acidic transcriptional activation domain(for a review see Schornack S, et al., (2006) J Plant Physiol 163(3):256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al., (2007) Appl and Enviro Micro 73(13): 4379-4384).These genes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas. See, e.g., U.S. Patent Publication No.20110301073, incorporated by reference in its entirety.

Specificity of these TAL effectors depends on the sequences found in thetandem repeats. The repeated sequence comprises approximately 102 bp andthe repeats are typically 91-100% homologous with each other (Bonas etal., ibid). Polymorphism of the repeats is usually located at positions12 and 13 and there appears to be a one-to-one correspondence betweenthe identity of the hypervariable diresidues at positions 12 and 13 withthe identity of the contiguous nucleotides in the TAL-effector's targetsequence (see Moscou and Bogdanove, (2009) Science 326:1501 and Boch etal., (2009) Science 326:1509-1512). Experimentally, the natural code forDNA recognition of these TAL-effectors has been determined such that anHD sequence at positions 12 and 13 leads to a binding to cytosine (C),NG binds to T, NI to A, C, G or T, NN binds to A or G, and ING binds toT. These DNA binding repeats have been assembled into proteins with newcombinations and numbers of repeats, to make artificial transcriptionfactors that are able to interact with new sequences and activate theexpression of a non-endogenous reporter gene in plant cells (Boch etal., ibid). Engineered TAL proteins have been linked to a FokI cleavagehalf domain to yield a TAL effector domain nuclease fusion (TALEN)exhibiting activity in a yeast reporter assay (plasmid based target).

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and Archaea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer.” Cas9 cleaves the DNA togenerate blunt ends at the double-stranded break (DSB) at sitesspecified by a 20-nucleotide guide sequence contained within the crRNAtranscript. Cas9 requires both the crRNA and the tracrRNA for sitespecific DNA recognition and cleavage. This system has now beenengineered such that the crRNA and tracrRNA can be combined into onemolecule (the “single guide RNA”), and the crRNA equivalent portion ofthe single guide RNA can be engineered to guide the Cas9 nuclease totarget any desired sequence (see Jinek et al., (2012) Science 337, pp.816-821, Jinek et al., (2013), eLife 2:e00471, and David Segal, (2013)eLife 2:e00563). Thus, the CRISPR/Cas system can be engineered to createa DSB at a desired target in a genome, and repair of the DSB can beinfluenced by the use of repair inhibitors to cause an increase in errorprone repair.

In other examples, the DNA binding transgene is a site specific nucleasethat comprises an engineered (non-naturally occurring) Meganuclease(also described as a homing endonuclease). The recognition sequences ofhoming endonucleases or meganucleases such as I-SceI, I-CeuI, PI-PspI,PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII, I-CreI,I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. Nos.5,420,032; 6,833,252; Belfort et al., (1997) Nucleic Acids Res.25:3379-30 3388; Dujon et al., (1989) Gene 82:115-118; Perler et al.,(1994) Nucleic Acids Res. 22, 11127; Jasin (1996) Trends Genet.12:224-228; Gimble et al., (1996) J. Mol. Biol. 263:163-180; Argast etal., (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue. In addition, the DNA-binding specificity of homingendonucleases and meganucleases can be engineered to bind non-naturaltarget sites. See, for example, Chevalier et al., (2002) Molec. Cell10:895-905; Epinat et al., (2003) Nucleic Acids Res. 5 31:2952-2962;Ashworth et al., (2006) Nature 441:656-659; Paques et al., (2007)Current Gene Therapy 7:49-66; U.S. Patent Publication No. 20070117128.The DNA-binding domains of the homing endonucleases and meganucleasesmay be altered in the context of the nuclease as a whole (i.e., suchthat the nuclease includes the cognate cleavage domain) or may be fusedto a heterologous cleavage domain.

As used herein, the term “transformation” encompasses all techniquesthat a nucleic acid molecule can be introduced into such a cell.Examples include, but are not limited to: transfection with viralvectors; transformation with plasmid vectors; electroporation;lipofection; microinjection (Mueller et al., (1978) Cell 15:579-85);Agrobacterium-mediated transfer; direct DNA uptake; WHISKERS™-mediatedtransformation; and microprojectile bombardment. These techniques may beused for both stable transformation and transient transformation of aplant cell. “Stable transformation” refers to the introduction of anucleic acid fragment into a genome of a host organism resulting ingenetically stable inheritance. Once stably transformed, the nucleicacid fragment is stably integrated in the genome of the host organismand any subsequent generation. Host organisms containing the transformednucleic acid fragments are referred to as “transgenic” organisms.“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

An exogenous nucleic acid sequence. In one example, a transgene is agene sequence (e.g., an herbicide-resistance gene), a gene encoding anindustrially or pharmaceutically useful compound, or a gene encoding adesirable agricultural trait. In yet another example, the transgene isan antisense nucleic acid sequence, wherein expression of the antisensenucleic acid sequence inhibits expression of a target nucleic acidsequence. A transgene may contain regulatory sequences operably linkedto the transgene (e.g., a promoter). In some embodiments, apolynucleotide sequence of interest is a transgene. However, in otherembodiments, a polynucleotide sequence of interest is an endogenousnucleic acid sequence, wherein additional genomic copies of theendogenous nucleic acid sequence are desired, or a nucleic acid sequencethat is in the antisense orientation with respect to the sequence of atarget nucleic acid molecule in the host organism.

As used herein, the term a transgenic “event” is produced bytransformation of plant cells with heterologous DNA, i.e., a nucleicacid construct that includes a transgene of interest, regeneration of apopulation of plants resulting from the insertion of the transgene intothe genome of the plant, and selection of a particular plantcharacterized by insertion into a particular genome location. The term“event” refers to the original transformant and progeny of thetransformant that include the heterologous DNA. The term “event” alsorefers to progeny produced by a sexual outcross between the transformantand another variety that includes the genomic/transgene DNA. Even afterrepeated back-crossing to a recurrent parent, the inserted transgene DNAand flanking genomic DNA (genomic/transgene DNA) from the transformedparent is present in the progeny of the cross at the same chromosomallocation. The term “event” also refers to DNA from the originaltransformant and progeny thereof comprising the inserted DNA andflanking genomic sequence immediately adjacent to the inserted DNA thatwould be expected to be transferred to a progeny that receives insertedDNA including the transgene of interest as the result of a sexual crossof one parental line that includes the inserted DNA (e.g., the originaltransformant and progeny resulting from selfing) and a parental linethat does not contain the inserted DNA.

As used herein, the terms “Polymerase Chain Reaction” or “PCR” define aprocedure or technique in which minute amounts of nucleic acid, RNAand/or DNA, are amplified as described in U.S. Pat. No. 4,683,195 issuedJul. 28, 1987. Generally, sequence information from the ends of theregion of interest or beyond needs to be available, such thatoligonucleotide primers can be designed; these primers will be identicalor similar in sequence to opposite strands of the template to beamplified. The 5′ terminal nucleotides of the two primers may coincidewith the ends of the amplified material. PCR can be used to amplifyspecific RNA sequences, specific DNA sequences from total genomic DNA,and cDNA transcribed from total cellular RNA, bacteriophage or plasmidsequences, etc. See generally Mullis et al., Cold Spring Harbor Symp.Quant. Biol., 51:263 (1987); Erlich, ed., PCR Technology, (StocktonPress, NY, 1989).

As used herein, the term “primer” refers to an oligonucleotide capableof acting as a point of initiation of synthesis along a complementarystrand when conditions are suitable for synthesis of a primer extensionproduct. The synthesizing conditions include the presence of fourdifferent deoxyribonucleotide triphosphates and at least onepolymerization-inducing agent such as reverse transcriptase or DNApolymerase. These are present in a suitable buffer, which may includeconstituents which are co-factors or which affect conditions such as pHand the like at various suitable temperatures. A primer is preferably asingle strand sequence, such that amplification efficiency is optimized,but double stranded sequences can be utilized.

As used herein, the term “probe” refers to an oligonucleotide thathybridizes to a target sequence. In the TaqMan® or TaqMan®-style assayprocedure, the probe hybridizes to a portion of the target situatedbetween the annealing site of the two primers. A probe includes abouteight nucleotides, about ten nucleotides, about fifteen nucleotides,about twenty nucleotides, about thirty nucleotides, about fortynucleotides, or about fifty nucleotides. In some embodiments, a probeincludes from about eight nucleotides to about fifteen nucleotides. Aprobe can further include a detectable label, e.g., a fluorophore(Texas-Red®, Fluorescein isothiocyanate, etc.,). The detectable labelcan be covalently attached directly to the probe oligonucleotide, e.g.,located at the probe's 5′ end or at the probe's 3′ end. A probeincluding a fluorophore may also further include a quencher, e.g., BlackHole Quencher™, Iowa Black™ etc.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence. Type-2 restrictionenzymes recognize and cleave DNA at the same site, and include but arenot limited to XbaI, BamHI, HindIII, EcoRI, XhoI, SalI, KpnI, AvaI, PstIand SmaI.

As used herein, the term “vector” is used interchangeably with the terms“construct”, “cloning vector” and “expression vector” and means thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. A “non-viral vector” is intended to mean any vector that doesnot comprise a virus or retrovirus. In some embodiments a “vector” is asequence of DNA comprising at least one origin of DNA replication and atleast one selectable marker gene. Examples include, but are not limitedto, a plasmid, cosmid, bacteriophage, bacterial artificial chromosome(BAC), or virus that carries exogenous DNA into a cell. A vector canalso include one or more genes, antisense molecules, and/or selectablemarker genes and other genetic elements known in the art. A vector maytransduce, transform, or infect a cell, thereby causing the cell toexpress the nucleic acid molecules and/or proteins encoded by thevector. The term “plasmid” defines a circular strand of nucleic acidcapable of autosomal replication in either a prokaryotic or a eukaryotichost cell. The term includes nucleic acid which may be either DNA or RNAand may be single- or double-stranded. The plasmid of the definition mayalso include the sequences which correspond to a bacterial origin ofreplication.

As used herein, the term “selectable marker gene” as used herein definesa gene or other expression cassette which encodes a protein whichfacilitates identification of cells into which the selectable markergene is inserted. For example a “selectable marker gene” encompassesreporter genes as well as genes used in plant transformation to, forexample, protect plant cells from a selective agent or provideresistance/tolerance to a selective agent. In one embodiment only thosecells or plants that receive a functional selectable marker are capableof dividing or growing under conditions having a selective agent.Examples of selective agents can include, for example, antibiotics,including spectinomycin, neomycin, kanamycin, paromomycin, gentamicin,and hygromycin. These selectable markers include neomycinphosphotransferase (npt II), which expresses an enzyme conferringresistance to the antibiotic kanamycin, and genes for the relatedantibiotics neomycin, paromomycin, gentamicin, and G418, or the gene forhygromycin phosphotransferase (hpt), which expresses an enzymeconferring resistance to hygromycin. Other selectable marker genes caninclude genes encoding herbicide resistance including bar or pat(resistance against glufosinate ammonium or phosphinothricin),acetolactate synthase (ALS, resistance against inhibitors such assulfonylureas (SUs), imidazolinones (IMIs), triazolopyrimidines (TPs),pyrimidinyl oxybenzoates (POBs), and sulfonylamino carbonyltriazolinones that prevent the first step in the synthesis of thebranched-chain amino acids), glyphosate, 2,4-D, and metal resistance orsensitivity. Examples of “reporter genes” that can be used as aselectable marker gene include the visual observation of expressedreporter gene proteins such as proteins encoding β-glucuronidase (GUS),luciferase, green fluorescent protein (GFP), yellow fluorescent protein(YFP), DsRed, β-galactosidase, chloramphenicol acetyltransferase (CAT),alkaline phosphatase, and the like. The phrase “marker-positive” refersto plants that have been transformed to include a selectable markergene.

As used herein, the term “detectable marker” refers to a label capableof detection, such as, for example, a radioisotope, fluorescentcompound, bioluminescent compound, a chemiluminescent compound, metalchelator, or enzyme. Examples of detectable markers include, but are notlimited to, the following: fluorescent labels (e.g., FITC, rhodamine,lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). In anembodiment, a detectable marker can be attached by spacer arms ofvarious lengths to reduce potential steric hindrance.

As used herein, the terms “cassette”, “expression cassette” and “geneexpression cassette” refer to a segment of DNA that can be inserted intoa nucleic acid or polynucleotide at specific restriction sites or byhomologous recombination. As used herein the segment of DNA comprises apolynucleotide that encodes a polypeptide of interest, and the cassetteand restriction sites are designed to ensure insertion of the cassettein the proper reading frame for transcription and translation. In anembodiment, an expression cassette can include a polynucleotide thatencodes a polypeptide of interest and having elements in addition to thepolynucleotide that facilitate transformation of a particular host cell.In an embodiment, a gene expression cassette may also include elementsthat allow for enhanced expression of a polynucleotide encoding apolypeptide of interest in a host cell. These elements may include, butare not limited to: a promoter, a minimal promoter, an enhancer, aresponse element, a terminator sequence, a polyadenylation sequence, andthe like.

As used herein a “linker” or “spacer” is a bond, molecule or group ofmolecules that binds two separate entities to one another. Linkers andspacers may provide for optimal spacing of the two entities or mayfurther supply a labile linkage that allows the two entities to beseparated from each other. Labile linkages include photocleavablegroups, acid-labile moieties, base-labile moieties and enzyme-cleavablegroups. The terms “polylinker” or “multiple cloning site” as used hereindefines a cluster of three or more Type-2 restriction enzyme siteslocated within 10 nucleotides of one another on a nucleic acid sequence.Constructs comprising a polylinker are utilized for the insertion and/orexcision of nucleic acid sequences such as the coding region of a gene.In other instances the term “polylinker” as used herein refers to astretch of nucleotides that are targeted for joining two sequences viaany known seamless cloning method (i.e., Gibson Assembly®, NEBuilderHiFiDNA Assembly®, Golden Gate Assembly®, BioBrick® Assembly, etc.).Constructs comprising a polylinker areutilized for the insertion and/orexcision of nucleic acid sequences such as the coding region of a gene.

As used herein, the term “control” refers to a sample used in ananalytical procedure for comparison purposes. A control can be“positive” or “negative”. For example, where the purpose of ananalytical procedure is to detect a differentially expressed transcriptor polypeptide in cells or tissue, it is generally preferable to includea positive control, such as a sample from a known plant exhibiting thedesired expression, and a negative control, such as a sample from aknown plant lacking the desired expression.

As used herein, the term “plant” includes a whole plant and anydescendant, cell, tissue, or part of a plant. A class of plant that canbe used in the present invention is generally as broad as the class ofhigher and lower plants amenable to mutagenesis including angiosperms(monocotyledonous and dicotyledonous plants), gymnosperms, ferns andmulticellular algae. Thus, “plant” includes dicot and monocot plants.The term “plant parts” include any part(s) of a plant, including, forexample and without limitation: seed (including mature seed and immatureseed); a plant cutting; a plant cell; a plant cell culture; a plantorgan (e.g., pollen, embryos, flowers, fruits, shoots, leaves, roots,stems, and explants). A plant tissue or plant organ may be a seed,protoplast, callus, or any other group of plant cells that is organizedinto a structural or functional unit. A plant cell or tissue culture maybe capable of regenerating a plant having the physiological andmorphological characteristics of the plant from which the cell or tissuewas obtained, and of regenerating a plant having substantially the samegenotype as the plant. In contrast, some plant cells are not capable ofbeing regenerated to produce plants. Regenerable cells in a plant cellor tissue culture may be embryos, protoplasts, meristematic cells,callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks, or stalks.

Plant parts include harvestable parts and parts useful for propagationof progeny plants. Plant parts useful for propagation include, forexample and without limitation: seed; fruit; a cutting; a seedling; atuber; and a rootstock. A harvestable part of a plant may be any usefulpart of a plant, including, for example and without limitation: flower;pollen; seedling; tuber; leaf; stem; fruit; seed; and root.

A plant cell is the structural and physiological unit of the plant,comprising a protoplast and a cell wall. A plant cell may be in the formof an isolated single cell, or an aggregate of cells (e.g., a friablecallus and a cultured cell), and may be part of a higher organized unit(e.g., a plant tissue, plant organ, and plant). Thus, a plant cell maybe a protoplast, a gamete producing cell, or a cell or collection ofcells that can regenerate into a whole plant. As such, a seed, whichcomprises multiple plant cells and is capable of regenerating into awhole plant, is considered a “plant cell” in embodiments herein.

As used herein, the term “small RNA” refers to several classes ofnon-coding ribonucleic acid (ncRNA). The term small RNA describes theshort chains of ncRNA produced in bacterial cells, animals, plants, andfungi. These short chains of ncRNA may be produced naturally within thecell or may be produced by the introduction of an exogenous sequencethat expresses the short chain or ncRNA. The small RNA sequences do notdirectly code for a protein, and differ in function from other RNA inthat small RNA sequences are only transcribed and not translated. Thesmall RNA sequences are involved in other cellular functions, includinggene expression and modification. Small RNA molecules are usually madeup of about 20 to 30 nucleotides. The small RNA sequences may be derivedfrom longer precursors. The precursors form structures that fold back oneach other in self-complementary regions; they are then processed by thenuclease Dicer in animals or DCL1 in plants.

Many types of small RNA exist either naturally or produced artificially,including microRNAs (miRNAs), short interfering RNAs (siRNAs), antisenseRNA, short hairpin RNA (shRNA), and small nucleolar RNAs (snoRNAs).Certain types of small RNA, such as microRNA and siRNA, are important ingene silencing and RNA interference (RNAi). Gene silencing is a processof genetic regulation in which a gene that would normally be expressedis “turned off” by an intracellular element, in this case, the smallRNA. The protein that would normally be formed by this geneticinformation is not formed due to interference, and the information codedin the gene is blocked from expression.

As used herein, the term “small RNA” encompasses RNA molecules describedin the literature as “tiny RNA” (Storz, (2002) Science 296:1260-3;Illangasekare et al., (1999) RNA 5:1482-1489); prokaryotic “small RNA”(sRNA) (Wassarman et al., (1999) Trends Microbiol. 7:37-45); eukaryotic“noncoding RNA (ncRNA)”; “micro-RNA (miRNA)”; “small non-mRNA (snmRNA)”;“functional RNA (fRNA)”; “transfer RNA (tRNA)”; “catalytic RNA” [e.g.,ribozymes, including self-acylating ribozymes (Illangaskare et al.,(1999) RNA 5:1482-1489); “small nucleolar RNAs (snoRNAs),” “tmRNA”(a.k.a. “10S RNA,” Muto et al., (1998) Trends Biochem Sci. 23:25-29; andGillet et al., (2001) Mol Microbiol. 42:879-885); RNAi moleculesincluding without limitation “small interfering RNA (siRNA),”“endoribonuclease-prepared siRNA (e-siRNA),” “short hairpin RNA(shRNA),” and “small temporally regulated RNA (stRNA),” “diced siRNA(d-siRNA),” and aptamers, oligonucleotides and other synthetic nucleicacids that comprise at least one uracil base.

Unless otherwise specifically explained, all technical and scientificterms used herein have the same meaning as commonly understood by thoseof ordinary skill in the art to which this disclosure belongs.Definitions of common terms in molecular biology can be found in, forexample: Lewin, Genes V, Oxford University Press, 1994 (ISBN0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of MolecularBiology, Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Meyers(ed.), Molecular Biology and Biotechnology: A Comprehensive DeskReference, VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

As used herein, the articles, “a,” “an,” and “the” include pluralreferences unless the context clearly and unambiguously dictatesotherwise.

III. Brachypodium distachyon Metallothionein-like Gene (Mtl) Promoterand Nucleic Acids Comprising the Same

Provided are methods and compositions for using a promoter from aBrachypodium distachyon metallothionein-like gene (mtl) to express nonmetallothionein-like transgenes in plant. In an embodiment, a promotercan be the Brachypodium distachyon metallothionein-like gene (mtl)promoter of SEQ ID NO:1.

In an embodiment, a polynucleotide is provided comprising a promoter,wherein the promoter is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100% identical to SEQ ID NO:1. Inan embodiment, a promoter is a Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising a polynucleotide ofat least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,99.5%, 99.8%, or 100% identity to the polynucleotide of SEQ ID NO:1. Inan embodiment, an isolated polynucleotide is provided comprising atleast 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.8%, or 100% identity to the polynucleotide of SEQ ID NO:1. In anembodiment, a nucleic acid vector is provided comprising a Brachypodiumdistachyon metallothionein-like gene (mtl) promoter of SEQ ID NO: 1. Inan embodiment, a polynucleotide is provided comprising a Brachypodiumdistachyon metallothionein-like gene (mtl) promoter that is operablylinked to a polylinker. In an embodiment, a gene expression cassette isprovided comprising a Brachypodium distachyon metallothionein-like gene(mtl) promoter that is operably linked to a non metallothionein-liketransgene. In an embodiment, a nucleic acid vector is providedcomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter that is operably linked to a non metallothionein-liketransgene. In one embodiment, the promoter consists of SEQ ID NO: 1. Inan illustrative embodiment, a nucleic acid vector comprises aBrachypodium distachyon metallothionein-like gene (mtl) promoter that isoperably linked to a transgene, wherein the transgene can be aninsecticidal resistance transgene, an herbicide tolerance transgene, anitrogen use efficiency transgene, a water use efficiency transgene, anutritional quality transgene, a DNA binding transgene, a small RNAtransgene, selectable marker transgene, or combinations thereof.

Transgene expression may also be regulated by the 3′-untranslated generegion (i.e., 3′-UTR) located downstream of the gene's coding sequence.Both a promoter and a 3′-UTR can regulate transgene expression. While apromoter is necessary to drive transcription, a 3′-UTR gene region canterminate transcription and initiate polyadenylation of a resulting mRNAtranscript for translation and protein synthesis. A 3′-UTR gene regionaids stable expression of a transgene.

In an embodiment, a nucleic acid vector is provided comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoter asdescribed herein and a 3′-UTR. In an embodiment, the nucleic acid vectorcomprises a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR. In an embodiment, the Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR is SEQ ID NO:3.

In an embodiment, a nucleic acid vector is provided comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoter asdescribed herein and a 3′-UTR, wherein the 3′-UTR is at least 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8%, or 100%identical to the polynucleotide of SEQ ID NO:3. In an embodiment, anucleic acid vector is provided comprising a Brachypodium distachyonmetallothionein-like gene (mtl) promoter as described herein and the3′-UTR wherein the Brachypodium distachyon metallothionein-like gene(mtl) promoter and 3′-UTR are both operably linked to opposite ends of apolylinker. In an embodiment, a gene expression cassette is providedcomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter as described herein and a 3′-UTR, wherein the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter and 3′-UTR are bothoperably linked to opposite ends of a non metallothionein-liketransgene. In one embodiment the 3′-UTR, consists of SEQ ID NO:3. In oneembodiment, a gene expression cassette is provided comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoter asdescribed herein and a 3′-UTR, wherein the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprises SEQ ID NO: 1 and the3′-UTR comprises SEQ ID NO: 3 wherein the promoter and 3′-UTR areoperably linked to opposite ends of a non metallothionein-liketransgene. In an aspect of this embodiment the 3′-UTR, consists of SEQID NO:3. In another aspect of this embodiment the promoter consists ofSEQ ID NO: 1. In an illustrative embodiment, a gene expression cassettecomprises a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR that is operably linked to a transgene, wherein the transgene canbe an insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, asmall RNA transgene, a selectable marker transgene, or combinationsthereof. In a further embodiment the transgene is operably linked to aBrachypodium distachyon metallothionein-like gene (mtl) promoter and a3′-UTR from the same metallothionein-like gene.

Transgene expression may also be regulated by an intron region locateddownstream of the promoter sequence. Both a promoter and an intron canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of an intron can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. Anintron gene region aids stable expression of a transgene. In a furtherembodiment an intron is operably linked to a Brachypodium distachyonmetallothionein-like gene (mtl) promoter.

Transgene expression may also be regulated by a 5′-UTR region locateddownstream of the promoter sequence. Both a promoter and a 5′-UTR canregulate transgene expression. While a promoter is necessary to drivetranscription, the presence of a 5′-UTR can increase expression levelsresulting in mRNA transcript for translation and protein synthesis. A5′-UTR gene region aids stable expression of a transgene. In a furtherembodiment an 5′-UTR is operably linked to a Brachypodium distachyonmetallothionein-like gene (mtl) promoter.

A Brachypodium distachyon metallothionein-like gene (mtl) promoter mayalso comprise one or more additional sequence elements. In someembodiments, a Brachypodium distachyon metallothionein-like gene (mtl)promoter may comprise an exon (e.g., a leader or signal peptide such asa chloroplast transit peptide or ER retention signal). For example andwithout limitation, a Brachypodium distachyon metallothionein-like gene(mtl) promoter may encode an exon incorporated into the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter as a furtherembodiment.

In an embodiment, a nucleic acid vector comprises a gene expressioncassette as disclosed herein. In an embodiment, a vector can be aplasmid, a cosmid, a bacterial artificial chromosome (BAC), abacteriophage, a virus, or an excised polynucleotide fragment for use indirect transformation or gene targeting such as a donor DNA.

In accordance with one embodiment a nucleic acid vector is providedcomprising a recombinant gene expression cassette wherein therecombinant gene expression cassette comprises a Brachypodium distachyonmetallothionein-like gene (mtl) promoter operably linked to a polylinkersequence, a non metallothionein-like transgene or combination thereof.In one embodiment the recombinant gene cassette comprises a Brachypodiumdistachyon metallothionein-like gene (mtl) promoter operably linked to anon metallothionein-like transgene. In one embodiment the recombinantgene cassette comprises a Brachypodium distachyon metallothionein-likegene (mtl) promoter as disclosed herein is operably linked to apolylinker sequence. The polylinker is operably linked to theBrachypodium distachyon metallothionein-like gene (mtl) promoter in amanner such that insertion of a coding sequence into one of therestriction sites of the polylinker will operably link the codingsequence allowing for expression of the coding sequence when the vectoris transformed or transfected into a host cell.

In accordance with one embodiment the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprises SEQ ID NO: 1 or asequence that has at least 80, 85, 90, 95 or 99% sequence identity withSEQ ID NO: 1. In accordance with one embodiment the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter comprises SEQ ID NO:1 or a sequence that has 80, 85, 90, 95 or 99% sequence identity withSEQ ID NO: 1. In accordance with one embodiment the promoter sequencehas a total length of no more than 1.5, 2, 2.5, 3 or 4 kb. In accordancewith one embodiment the Brachypodium distachyon metallothionein-likegene (mtl) promoter consists of SEQ ID NO: 1 or a 2,000 bp sequence thathas at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 1.In accordance with one embodiment the Brachypodium distachyonmetallothionein-like gene (mtl) promoter consists of SEQ ID NO: 1 or a2,000 bp sequence that has 80, 85, 90, 95 or 99% sequence identity withSEQ ID NO: 1.

Further provided as an embodiment of the Brachypodium distachyonmetallothionein-like gene (mtl) promoter is SEQ ID NO:12. This modifiedBrachypodium distachyon metallothionein-like gene (mtl) promotersequence can be used herein to drive the expression of a transgene, andprovide alternative promoter sequences that may be used instead of SEQID NO:1 as provided by the subject disclosure herein. The promoterpolynucleotide sequences of SEQ ID NO:1 and SEQ ID NO:12 share 97.2%sequence identity. The promoter of SEQ ID NO:12 is provided as anadditional embodiment of a Brachypodium distachyon metallothionein-likegene (mtl) promoter.

Also provided as embodiments of the Brachypodium distachyonmetallothionein-like gene (mtl) promoter are SEQ ID NO:7 (1,730 bp), SEQID NO:8 (1,530 bp), SEQ ID NO:9 (1,330 bp), SEQ ID NO:10 (1,130 bp) andSEQ ID NO:11 (1,001 bp) as shown in FIG. 2. These truncated Brachypodiumdistachyon metallothionein-like gene (mtl) promoter sequences can beused herein to drive the expression of a transgene, and providealternative promoter sequences that may be used instead of SEQ ID NO:1as provided by the subject disclosure herein. The 1,730 bp promoter ofSEQ ID NO:7 is provided as an additional embodiment of a Brachypodiumdistachyon metallothionein-like gene (mtl) promoter. In addition, the1,530 bp promoter of SEQ ID NO:8 is provided as an additional embodimentof a Brachypodium distachyon metallothionein-like gene (mtl) promoter.The 1,330 bp promoter of SEQ ID NO:9 is provided as an additionalembodiment of a Brachypodium distachyon metallothionein-like gene (mtl)promoter. Further, the 1,130 bp promoter of SEQ ID NO:10 is provided asan additional embodiment of a Brachypodium distachyonmetallothionein-like gene (mtl) promoter. Furthermore, the 1,001 bppromoter of SEQ ID NO:9 is provided as an additional embodiment of aBrachypodium distachyon metallothionein-like gene (mtl) promoter.

In accordance with one embodiment a nucleic acid vector is providedcomprising a gene cassette that consists of a Brachypodium distachyonmetallothionein-like gene (mtl) promoter, a non metallothionein-liketransgene and a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR of SEQ ID NO: 3. In an embodiment, the Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR of SEQ ID NO: 3 is operablylinked to the 3′ end of the non metallothionein-like transgene. In afurther embodiment the 3′ untranslated sequence comprises SEQ ID NO: 3or a sequence that has at least 80, 85, 90, 95, 99 or 100% sequenceidentity with SEQ ID NO: 3. In a further embodiment the 3′ untranslatedsequence comprises SEQ ID NO: 3 or a sequence that has 80, 85, 90, 95,99 or 100% sequence identity with SEQ ID NO: 3. In accordance with oneembodiment a nucleic acid vector is provided comprising a gene cassettethat consists of SEQ ID NO: 1, or a 2,000 bp sequence that has at least80, 85, 90, 95, or 99% sequence identity with SEQ ID NO: 1, a nonmetallothionein-like transgene and a 3′-UTR, wherein SEQ ID NO: 1 isoperably linked to the 5′ end of the non metallothionein-like transgeneand the 3′-UTR of SEQ ID NO:3 is operably linked to the 3′ end of thenon metallothionein-like transgene. In accordance with one embodiment anucleic acid vector is provided comprising a gene cassette that consistsof SEQ ID NO: 1, or a 2,000 bp sequence that has 80, 85, 90, 95, or 99%sequence identity with SEQ ID NO: 1, a non metallothionein-liketransgene and a 3′-UTR, wherein SEQ ID NO: 1 is operably linked to the5′ end of the non metallothionein-like transgene and the 3′-UTR of SEQID NO:3 is operably linked to the 3′ end of the non metallothionein-liketransgene. In a further embodiment the 3′ untranslated sequencecomprises SEQ ID NO: 3 or a sequence that has at least 80, 85, 90, 95,99 or 100% sequence identity with SEQ ID NO: 3. In a further embodimentthe 3′ untranslated sequence consists of SEQ ID NO: 3, or a 1,002 bpsequence that has at least 80, 85, 90, 95, or 99% sequence identity withSEQ ID NO: 3. In a further embodiment the 3′ untranslated sequencecomprises SEQ ID NO: 3 or a sequence that has 80, 85, 90, 95, 99 or 100%sequence identity with SEQ ID NO: 3. In a further embodiment the 3′untranslated sequence consists of SEQ ID NO: 3, or a 1,002 bp sequencethat has 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO: 3.

Further provided as embodiments of the Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR is SEQ ID NO:17 and SEQ ID NO:18.These modified Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR sequences can be used herein to terminate the expression of atransgene, and provide alternative 3′-UTR sequences that may be usedinstead of SEQ ID NO:3 as provided by the subject disclosure herein. The3′-UTR polynucleotide sequences of SEQ ID NO:3 and SEQ ID NO:17 share94.2% sequence identity. The 3′-UTR polynucleotide sequences of SEQ IDNO:3 and SEQ ID NO:18 share 97.5% sequence identity. Finally, the 3′-UTRpolynucleotide sequences of SEQ ID NO:18 and SEQ ID NO:17 share 92.7%sequence identity. The 3′-UTR of SEQ ID NO:17 is provided as anadditional embodiment of a Brachypodium distachyon metallothionein-likegene (mtl) 3′-UTR. The 3′-UTR of SEQ ID NO:18 is provided as anadditional embodiment of a Brachypodium distachyon metallothionein-likegene (mtl) 3′-UTR.

Also provided as embodiments of the Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR are SEQ ID NO:13 (264 bp), SEQ IDNO:14 (332 bp), SEQ ID NO:15 (630 bp), and SEQ ID NO:16 (727 bp) asshown in FIG. 4. These truncated Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR sequences can be used herein toterminate the expression of a transgene, and provide alternative 3′-UTRsequences that may be used instead of SEQ ID NO:3 as provided by thesubject disclosure herein. The 264 bp 3′-UTR of SEQ ID NO:13 is providedas an additional embodiment of a Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR. In addition, the 332 bp 3′-UTRof SEQ ID NO:14 is provided as an additional embodiment of aBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR. The 630bp 3′-UTR of SEQ ID NO:15 is provided as an additional embodiment of aBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR. Further,the 727 bp 3′-UTR of SEQ ID NO:16 is provided as an additionalembodiment of a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR.

In one embodiment a nucleic acid construct is provided comprising apromoter and a non metallothionein-like transgene and optionally one ormore of the following elements:

-   -   a) a 5′ untranslated region;    -   b) an intron; and    -   c) a 3′ untranslated region,        wherein,    -   the promoter consists of SEQ ID NO:1 or a sequence having 98%        sequence identity with SEQ ID NO:1; and    -   the 3′ untranslated region consists of SEQ ID NO:3 or a sequence        having 98% sequence identity with SEQ ID NO:3; further wherein        said promoter is operably linked to said transgene and each        optional element, when present, is also operably linked to both        the promoter and the transgene. In a further embodiment a        transgenic cell is provided comprising the nucleic acid        construct disclosed immediately above. In one embodiment the        transgenic cell is a plant cell, and in a further embodiment a        plant is provided wherein the plant comprises said transgenic        cells.

In one embodiment a nucleic acid construct is provided comprising apromoter and a non metallothionein-like transgene and optionally one ormore of the following elements:

-   -   a) a 3′ untranslated region,        wherein,    -   the promoter consists of SEQ ID NO:1 or a sequence having 98%        sequence identity with SEQ ID NO:1; and    -   the 3′ untranslated region consists of SEQ ID NO:3 or a sequence        having 98% sequence identity with SEQ ID NO:3; further wherein        said promoter is operably linked to said transgene and each        optional element, when present, is also operably linked to both        the promoter and the transgene. In a further embodiment a        transgenic cell is provided comprising the nucleic acid        construct disclosed immediately above. In one embodiment the        transgenic cell is a plant cell, and in a further embodiment a        plant is provided wherein the plant comprises said transgenic        cells.

In one embodiment a nucleic acid construct is provided comprising apromoter and a polylinker and optionally one or more of the followingelements:

-   -   a) a ′5 untranslated region,    -   b) an intron, and    -   c) a 3′ untranslated region,        wherein,    -   the promoter consists of SEQ ID NO:1 or a sequence having 98%        sequence identity with SEQ ID NO:1; and    -   the 3′ untranslated region consists of SEQ ID NO:3 or a sequence        having 98% sequence identity with SEQ ID NO:3; further wherein        said promoter is operably linked to said polylinker and each        optional element, when present, is also operably linked to both        the promoter and the polylinker.

In accordance with one embodiment the nucleic acid vector furthercomprises a sequence encoding a selectable maker. In accordance with oneembodiment the recombinant gene cassette is operably linked to anAgrobacterium T-DNA border. In accordance with one embodiment therecombinant gene cassette further comprises a first and second T-DNAborder, wherein the first T-DNA border is operably linked to one end ofthe gene construct, and the second T-DNA border is operably linked tothe other end of the gene construct. The first and second AgrobacteriumT-DNA borders can be independently selected from T-DNA border sequencesoriginating from bacterial strains selected from the group consisting ofa nopaline synthesizing Agrobacterium T-DNA border, an ocotopinesynthesizing Agrobacterium T-DNA border, a mannopine synthesizingAgrobacterium T-DNA border, a succinamopine synthesizing AgrobacteriumT-DNA border, or any combination thereof. In one embodiment anAgrobacterium strain selected from the group consisting of a nopalinesynthesizing strain, a mannopine synthesizing strain, a succinamopinesynthesizing strain, or an octopine synthesizing strain is provided,wherein said strain comprises a plasmid wherein the plasmid comprises atransgene operably linked to a sequence selected from SEQ ID NO: 1 or asequence having 80, 85, 90, 95, or 99% sequence identity with SEQ ID NO:1.

Transgenes of interest that are suitable for use in the presentdisclosed constructs include, but are not limited to, coding sequencesthat confer (1) resistance to pests or disease, (2) tolerance toherbicides, (3) value added agronomic traits, such as; yieldimprovement, nitrogen use efficiency, water use efficiency, andnutritional quality, (4) binding of a protein to DNA in a site specificmanner, (5) expression of small RNA, and (6) selectable markers. Inaccordance with one embodiment, the transgene encodes a selectablemarker or a gene product conferring insecticidal resistance, herbicidetolerance, small RNA expression, nitrogen use efficiency, water useefficiency, or nutritional quality.

1. Insect Resistance

Various insect resistance coding sequences can be operably linked to theBrachypodium distachyon metallothionein-like gene (mtl) promotercomprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 1. Various insect resistancecoding sequences can be operably linked to the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising SEQ ID NO: 1, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 1. The operably linked sequences can then be incorporated into achosen vector to allow for identification and selection of transformedplants (“transformants”). Exemplary insect resistance coding sequencesare known in the art. As embodiments of insect resistance codingsequences that can be operably linked to the regulatory elements of thesubject disclosure, the following traits are provided. Coding sequencesthat provide exemplary Lepidopteran insect resistance include: cry1A;cry1A.105; cry1Ab; cry1Ab(truncated); cry1Ab-Ac (fusion protein); cry1Ac(marketed as Widestrike®); cry1C; cry1F (marketed as Widestrike®);cry1Fa2; cry2Ab2; cry2Ae; cry9C; mocry1F; pinII (protease inhibitorprotein); vip3A(a); and vip3Aa20. Coding sequences that provideexemplary Coleopteran insect resistance include: cry34Ab1 (marketed asHerculex®); cry35Ab1 (marketed as Herculex®); cry3A; cry3Bb1; dvsnf7;and mcry3A. Coding sequences that provide exemplary multi-insectresistance include ecry31.Ab. The above list of insect resistance genesis not meant to be limiting. Any insect resistance genes are encompassedby the present disclosure.

2. Herbicide Tolerance

Various herbicide tolerance coding sequences can be operably linked tothe Brachypodium distachyon metallothionein-like gene (mtl) promotercomprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 1. Various herbicide tolerancecoding sequences can be operably linked to the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising SEQ ID NO: 1, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 1. The operably linked sequences can then be incorporated into achosen vector to allow for identification and selection of transformedplants (“transformants”). Exemplary herbicide tolerance coding sequencesare known in the art. As embodiments of herbicide tolerance codingsequences that can be operably linked to the regulatory elements of thesubject disclosure, the following traits are provided. The glyphosateherbicide contains a mode of action by inhibiting the EPSPS enzyme(5-enolpyruvylshikimate-3-phosphate synthase). This enzyme is involvedin the biosynthesis of aromatic amino acids that are essential forgrowth and development of plants. Various enzymatic mechanisms are knownin the art that can be utilized to inhibit this enzyme. The genes thatencode such enzymes can be operably linked to the gene regulatoryelements of the subject disclosure. In an embodiment, selectable markergenes include, but are not limited to genes encoding glyphosateresistance genes include: mutant EPSPS genes such as 2mEPSPS genes, cp4EPSPS genes, mEPSPS genes, dgt-28 genes; aroA genes; and glyphosatedegradation genes such as glyphosate acetyl transferase genes (gat) andglyphosate oxidase genes (gox). These traits are currently marketed asGly-Tol™, Optimum® GAT®, Agrisure® GT and Roundup Ready®. Resistancegenes for glufosinate and/or bialaphos compounds include dsm-2, bar andpat genes. The bar and pat traits are currently marketed asLibertyLink®. Also included are tolerance genes that provide resistanceto 2,4-D such as aad-1 genes (it should be noted that aad-1 genes havefurther activity on arloxyphenoxypropionate herbicides) and aad-12 genes(it should be noted that aad-12 genes have further activity onpyidyloxyacetate synthetic auxins). These traits are marketed as Enlist®crop protection technology. Resistance genes for ALS inhibitors(sulfonylureas, imidazolinones, triazolopyrimidines,pyrimidinylthiobenzoates, and sulfonylamino-carbonyl-triazolinones) areknown in the art. These resistance genes most commonly result from pointmutations to the ALS encoding gene sequence. Other ALS inhibitorresistance genes include hra genes, the csr1-2 genes, Sr-HrA genes, andsurB genes. Some of the traits are marketed under the tradenameClearfield®. Herbicides that inhibit HPPD include the pyrazolones suchas pyrazoxyfen, benzofenap, and topramezone; triketones such asmesotrione, sulcotrione, tembotrione, benzobicyclon; and diketonitrilessuch as isoxaflutole. These exemplary HPPD herbicides can be toleratedby known traits. Examples of HPPD inhibitors include hppdPF_W336 genes(for resistance to isoxaflutole) and avhppd-03 genes (for resistance tomeostrione). An example of oxynil herbicide tolerant traits include thebxn gene, which has been showed to impart resistance to theherbicide/antibiotic bromoxynil. Resistance genes for dicamba includethe dicamba monooxygenase gene (dmo) as disclosed in International PCTPublication No. WO 2008/105890. Resistance genes for PPO or PROTOXinhibitor type herbicides (e.g., acifluorfen, butafenacil, flupropazil,pentoxazone, carfentrazone, fluazolate, pyraflufen, aclonifen,azafenidin, flumioxazin, flumiclorac, bifenox, oxyfluorfen, lactofen,fomesafen, fluoroglycofen, and sulfentrazone) are known in the art.Exemplary genes conferring resistance to PPO include over expression ofa wild-type Arabidopsis thaliana PPO enzyme (Lermontova I and Grimm B,(2000) Overexpression of plastidic protoporphyrinogen IX oxidase leadsto resistance to the diphenyl-ether herbicide acifluorfen. Plant Physiol122:75-83.), the B. subtilis PPO gene (Li, X. and Nicholl D. 2005.Development of PPO inhibitor-resistant cultures and crops. Pest Manag.Sci. 61:277-285 and Choi K W, Han O, Lee H J, Yun Y C, Moon Y H, Kim MK, Kuk Y I, Han S U and Guh J O, (1998) Generation of resistance to thediphenyl ether herbicide, oxyfluorfen, via expression of the Bacillussubtilis protoporphyrinogen oxidase gene in transgenic tobacco plants.Biosci Biotechnol Biochem 62:558-560.) Resistance genes for pyridinoxyor phenoxy proprionic acids and cyclohexones include the ACCaseinhibitor-encoding genes (e.g., Acc1-S1, Acc1-S2 and Acc1-S3). Exemplarygenes conferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid include haloxyfop, diclofop, fenoxyprop,fluazifop, and quizalofop. Finally, herbicides can inhibitphotosynthesis, including triazine or benzonitrile are providedtolerance by psbA genes (tolerance to triazine), 1s+ genes (tolerance totriazine), and nitrilase genes (tolerance to benzonitrile). The abovelist of herbicide tolerance genes is not meant to be limiting. Anyherbicide tolerance genes are encompassed by the present disclosure.

3. Agronomic Traits

Various agronomic trait coding sequences can be operably linked to theBrachypodium distachyon metallothionein-like gene (mtl) promotercomprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 1. Various agronomic traitcoding sequences can be operably linked to the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising SEQ ID NO: 1, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 1. The operably linked sequences can then be incorporated into achosen vector to allow for identification and selection of transformedplants (“transformants”). Exemplary agronomic trait coding sequences areknown in the art. As embodiments of agronomic trait coding sequencesthat can be operably linked to the regulatory elements of the subjectdisclosure, the following traits are provided. Delayed fruit softeningas provided by the pg genes inhibit the production of polygalacturonaseenzyme responsible for the breakdown of pectin molecules in the cellwall, and thus causes delayed softening of the fruit. Further, delayedfruit ripening/senescence of acc genes act to suppress the normalexpression of the native acc synthase gene, resulting in reducedethylene production and delayed fruit ripening. Whereas, the accd genesmetabolize the precursor of the fruit ripening hormone ethylene,resulting in delayed fruit ripening. Alternatively, the sam-k genescause delayed ripening by reducing S-adenosylmethionine (SAM), asubstrate for ethylene production. Drought stress tolerance phenotypesas provided by cspB genes maintain normal cellular functions under waterstress conditions by preserving RNA stability and translation. Anotherexample includes the EcBetA genes that catalyze the production of theosmoprotectant compound glycine betaine conferring tolerance to waterstress. In addition, the RmBetA genes catalyze the production of theosmoprotectant compound glycine betaine conferring tolerance to waterstress. Photosynthesis and yield enhancement is provided with the bbx32gene that expresses a protein that interacts with one or more endogenoustranscription factors to regulate the plant's day/night physiologicalprocesses. Ethanol production can be increase by expression of theamy797E genes that encode a thermostable alpha-amylase enzyme thatenhances bioethanol production by increasing the thermostability ofamylase used in degrading starch. Finally, modified amino acidcompositions can result by the expression of the cordapA genes thatencode a dihydrodipicolinate synthase enzyme that increases theproduction of amino acid lysine. The above list of agronomic traitcoding sequences is not meant to be limiting. Any agronomic trait codingsequence is encompassed by the present disclosure.

4. DNA Binding Proteins

Various DNA binding protein coding sequences can be operably linked tothe Brachypodium distachyon metallothionein-like gene (mtl) promotercomprising SEQ ID NO: 1, or a sequence that has at least 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 1. Various DNA binding proteincoding sequences can be operably linked to the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising SEQ ID NO: 1, or asequence that has 80, 85, 90, 95 or 99% sequence identity with SEQ IDNO: 1. The operably linked sequences can then be incorporated into achosen vector to allow for identification and selectable of transformedplants (“transformants”). Exemplary DNA binding protein coding sequencesare known in the art. As embodiments of DNA binding protein codingsequences that can be operably linked to the regulatory elements of thesubject disclosure, the following types of DNA binding proteins caninclude; Zinc Fingers, Talens, CRISPRS, and meganucleases. The abovelist of DNA binding protein coding sequences is not meant to belimiting. Any DNA binding protein coding sequences is encompassed by thepresent disclosure.

5. Small RNA

Various small RNAs can be operably linked to the Brachypodium distachyonmetallothionein-like gene (mtl) promoter comprising SEQ ID NO: 1, or asequence that has at least 80, 85, 90, 95 or 99% sequence identity withSEQ ID NO: 1. Various small RNAs can be operably linked to theBrachypodium distachyon metallothionein-like gene (mtl) promotercomprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95 or 99%sequence identity with SEQ ID NO: 1. The operably linked sequences canthen be incorporated into a chosen vector to allow for identificationand selection of transformed plants (“transformants”). Exemplary smallRNA traits are known in the art. As embodiments of small RNA codingsequences that can be operably linked to the regulatory elements of thesubject disclosure, the following traits are provided. For example,delayed fruit ripening/senescence of the anti-efe small RNA delaysripening by suppressing the production of ethylene via silencing of theACO gene that encodes an ethylene-forming enzyme. The altered ligninproduction of ccomt small RNA reduces content of guanacyl (G) lignin byinhibition of the endogenous S-adenosyl-L-methionine: trans-caffeoyl CoA3-O-methyltransferase (CCOMT gene). Further, the Black Spot BruiseTolerance in Solanum verrucosum can be reduced by the Ppo5 small RNAwhich triggers the degradation of Ppo5 transcripts to block black spotbruise development. Also included is the dvsnf7 small RNA that inhibitsWestern Corn Rootworm with dsRNA containing a 240 bp fragment of theWestern Corn Rootworm Snf7 gene. Modified starch/carbohydrates canresult from small RNA such as the pPhL small RNA (degrades PhLtranscripts to limit the formation of reducing sugars through starchdegradation) and pR1 small RNA (degrades R1 transcripts to limit theformation of reducing sugars through starch degradation). Additional,benefits such as reduced acrylamide resulting from the asn1 small RNAthat triggers degradation of Asn1 to impair asparagine formation andreduce polyacrylamide. Finally, the non-browning phenotype of pgas pposuppression small RNA results in suppressing PPO to produce apples witha non-browning phenotype. The above list of small RNAs is not meant tobe limiting. Any small RNA encoding sequences are encompassed by thepresent disclosure.

6. Selectable Markers

Various selectable markers also described as reporter genes can beoperably linked to the Brachypodium distachyon metallothionein-like gene(mtl) promoter comprising SEQ ID NO: 1, or a sequence that has at least80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 1. Variousselectable markers also described as reporter genes can be operablylinked to the Brachypodium distachyon metallothionein-like gene (mtl)promoter comprising SEQ ID NO: 1, or a sequence that has 80, 85, 90, 95or 99% sequence identity with SEQ ID NO: 1. The operably linkedsequences can then be incorporated into a chosen vector to allow foridentification and selectable of transformed plants (“transformants”).Many methods are available to confirm expression of selectable markersin transformed plants, including for example DNA sequencing and PCR(polymerase chain reaction), Southern blotting, RNA blotting,immunological methods for detection of a protein expressed from thevector. But, usually the reporter genes are observed through visualobservation of proteins that when expressed produce a colored product.Exemplary reporter genes are known in the art and encode β-glucuronidase(GUS), luciferase, green fluorescent protein (GFP), yellow fluorescentprotein (YFP, Phi-YFP), red fluorescent protein (DsRFP, RFP, etc),β-galactosidase, and the like (See Sambrook, et al., Molecular Cloning:A Laboratory Manual, Third Edition, Cold Spring Harbor Press, N.Y.,2001, the content of which is incorporated herein by reference in itsentirety).

Selectable marker genes are utilized for selection of transformed cellsor tissues. Selectable marker genes include genes encoding antibioticresistance, such as those encoding neomycin phosphotransferase II (NEO),spectinomycin/streptinomycin resistance (AAD), and hygromycinphosphotransferase (HPT or HGR) as well as genes conferring resistanceto herbicidal compounds. Herbicide resistance genes generally code for amodified target protein insensitive to the herbicide or for an enzymethat degrades or detoxifies the herbicide in the plant before it canact. For example, resistance to glyphosate has been obtained by usinggenes coding for mutant target enzymes,5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Genes and mutantsfor EPSPS are well known, and further described below. Resistance toglufosinate ammonium, bromoxynil, and 2,4-dichlorophenoxyacetate (2,4-D)have been obtained by using bacterial genes encoding PAT or DSM-2, anitrilase, an AAD-1, or an AAD-12, each of which are examples ofproteins that detoxify their respective herbicides.

In an embodiment, herbicides can inhibit the growing point or meristem,including imidazolinone or sulfonylurea, and genes forresistance/tolerance of acetohydroxyacid synthase (AHAS) andacetolactate synthase (ALS) for these herbicides are well known.Glyphosate resistance genes include mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSPs) and dgt-28 genes(via the introduction of recombinant nucleic acids and/or various formsof in vivo mutagenesis of native EPSPs genes), aroA genes and glyphosateacetyl transferase (GAT) genes, respectively). Resistance genes forother phosphono compounds include bar and pat genes from Streptomycesspecies, including Streptomyces hygroscopicus and Streptomycesviridichromogenes, and pyridinoxy or phenoxy proprionic acids andcyclohexones (ACCase inhibitor-encoding genes). Exemplary genesconferring resistance to cyclohexanediones and/oraryloxyphenoxypropanoic acid (including haloxyfop, diclofop, fenoxyprop,fluazifop, quizalofop) include genes of acetyl coenzyme A carboxylase(ACCase); Acc1-S1, Acc1-S2 and Acc1-S3. In an embodiment, herbicides caninhibit photosynthesis, including triazine (psbA and 1s+ genes) orbenzonitrile (nitrilase gene). Furthermore, such selectable markers caninclude positive selection markers such as phosphomannose isomerase(PMI) enzyme.

In an embodiment, selectable marker genes include, but are not limitedto genes encoding: 2,4-D; neomycin phosphotransferase II; cyanamidehydratase; aspartate kinase; dihydrodipicolinate synthase; tryptophandecarboxylase; dihydrodipicolinate synthase and desensitized aspartatekinase; bar gene; tryptophan decarboxylase; neomycin phosphotransferase(NEO); hygromycin phosphotransferase (HPT or HYG); dihydrofolatereductase (DHFR); phosphinothricin acetyltransferase;2,2-dichloropropionic acid dehalogenase; acetohydroxyacid synthase;5-enolpyruvyl-shikimate-phosphate synthase (aroA); haloarylnitrilase;acetyl-coenzyme A carboxylase; dihydropteroate synthase (sul I); and 32kD photosystem II polypeptide (psbA). An embodiment also includesselectable marker genes encoding resistance to: chloramphenicol;methotrexate; hygromycin; spectinomycin; bromoxynil; glyphosate; andphosphinothricin. The above list of selectable marker genes is not meantto be limiting. Any reporter or selectable marker gene are encompassedby the present disclosure.

In some embodiments the coding sequences are synthesized for optimalexpression in a plant. For example, in an embodiment, a coding sequenceof a gene has been modified by codon optimization to enhance expressionin plants. An insecticidal resistance transgene, an herbicide tolerancetransgene, a nitrogen use efficiency transgene, a water use efficiencytransgene, a nutritional quality transgene, a DNA binding transgene, ora selectable marker transgene can be optimized for expression in aparticular plant species or alternatively can be modified for optimalexpression in dicotyledonous or monocotyledonous plants. Plant preferredcodons may be determined from the codons of highest frequency in theproteins expressed in the largest amount in the particular plant speciesof interest. In an embodiment, a coding sequence, gene, or transgene isdesigned to be expressed in plants at a higher level resulting in highertransformation efficiency. Methods for plant optimization of genes arewell known. Guidance regarding the optimization and production ofsynthetic DNA sequences can be found in, for example, WO2013016546,WO2011146524, WO1997013402, U.S. Pat. Nos. 6,166,302, and 5,380,831,herein incorporated by reference.

Transformation

Suitable methods for transformation of plants include any method bywhich DNA can be introduced into a cell, for example and withoutlimitation: electroporation (see, e.g., U.S. Pat. No. 5,384,253);micro-projectile bombardment (see, e.g., U.S. Pat. Nos. 5,015,580,5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865);Agrobacterium-mediated transformation (see, e.g., U.S. Pat. Nos.5,635,055, 5,824,877, 5,591,616; 5,981,840, and 6,384,301); andprotoplast transformation (see, e.g., U.S. Pat. No. 5,508,184).

A DNA construct may be introduced directly into the genomic DNA of theplant cell using techniques such as agitation with silicon carbidefibers (see, e.g., U.S. Pat. Nos. 5,302,523 and 5,464,765), or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-73). Alternatively, the DNA construct can beintroduced into the plant cell via nanoparticle transformation (see,e.g., US Patent Publication No. 20090104700, which is incorporatedherein by reference in its entirety).

In addition, gene transfer may be achieved using non-Agrobacteriumbacteria or viruses such as Rhizobium sp. NGR234, Sinorhizoboiummeliloti, Mesorhizobium loti, potato virus X, cauliflower mosaic virusand cassava vein mosaic virus and/or tobacco mosaic virus, See, e.g.,Chung et al. (2006) Trends Plant Sci. 11(1):1-4.

Through the application of transformation techniques, cells of virtuallyany plant species may be stably transformed, and these cells may bedeveloped into transgenic plants by well-known techniques. For example,techniques that may be particularly useful in the context of cottontransformation are described in U.S. Pat. Nos. 5,846,797, 5,159,135,5,004,863, and 6,624,344; techniques for transforming Brassica plants inparticular are described, for example, in U.S. Pat. No. 5,750,871;techniques for transforming soy bean are described, for example, in U.S.Pat. No. 6,384,301; and techniques for transforming maize are described,for example, in U.S. Pat. Nos. 7,060,876 and 5,591,616, andInternational PCT Publication WO 95/06722.

After effecting delivery of an exogenous nucleic acid to a recipientcell, a transformed cell is generally identified for further culturingand plant regeneration. In order to improve the ability to identifytransformants, one may desire to employ a selectable marker gene withthe transformation vector used to generate the transformant. In anillustrative embodiment, a transformed cell population can be assayed byexposing the cells to a selective agent or agents, or the cells can bescreened for the desired marker gene trait.

Cells that survive exposure to a selective agent, or cells that havebeen scored positive in a screening assay, may be cultured in media thatsupports regeneration of plants. In an embodiment, any suitable planttissue culture media may be modified by including further substances,such as growth regulators. Tissue may be maintained on a basic mediawith growth regulators until sufficient tissue is available to beginplant regeneration efforts, or following repeated rounds of manualselection, until the morphology of the tissue is suitable forregeneration (e.g., at least 2 weeks), then transferred to mediaconducive to shoot formation. Cultures are transferred periodicallyuntil sufficient shoot formation has occurred. Once shoots are formed,they are transferred to media conducive to root formation. Oncesufficient roots are formed, plants can be transferred to soil forfurther growth and maturity.

Molecular Confirmation

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection can be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells can also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, or gfp genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art. Molecularconfirmation methods that can be used to identify transgenic plants areknown to those with skill in the art. Several exemplary methods arefurther described below.

Molecular Beacons have been described for use in sequence detection.Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking genomic and insert DNA junction. The unique structure of theFRET probe results in it containing a secondary structure that keeps thefluorescent and quenching moieties in close proximity. The FRET probeand PCR primers (one primer in the insert DNA sequence and one in theflanking genomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Following successful PCR amplification,hybridization of the FRET probe(s) to the target sequence results in theremoval of the probe secondary structure and spatial separation of thefluorescent and quenching moieties. A fluorescent signal indicates thepresence of the flanking genomic/transgene insert sequence due tosuccessful amplification and hybridization. Such a molecular beaconassay for detection of as an amplification reaction is an embodiment ofthe subject disclosure.

Hydrolysis probe assay, otherwise known as TAQMAN® (Life Technologies,Foster City, Calif.), is a method of detecting and quantifying thepresence of a DNA sequence. Briefly, a FRET oligonucleotide probe isdesigned with one oligo within the transgene and one in the flankinggenomic sequence for event-specific detection. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankinggenomic sequence) are cycled in the presence of a thermostablepolymerase and dNTPs. Hybridization of the FRET probe results incleavage and release of the fluorescent moiety away from the quenchingmoiety on the FRET probe. A fluorescent signal indicates the presence ofthe flanking/transgene insert sequence due to successful amplificationand hybridization. Such a hydrolysis probe assay for detection of as anamplification reaction is an embodiment of the subject disclosure.

KASPar® assays are a method of detecting and quantifying the presence ofa DNA sequence. Briefly, the genomic DNA sample comprising theintegrated gene expression cassette polynucleotide is screened using apolymerase chain reaction (PCR) based assay known as a KASPar® assaysystem. The KASPar® assay used in the practice of the subject disclosurecan utilize a KASPar® PCR assay mixture which contains multiple primers.The primers used in the PCR assay mixture can comprise at least oneforward primers and at least one reverse primer. The forward primercontains a sequence corresponding to a specific region of the DNApolynucleotide, and the reverse primer contains a sequence correspondingto a specific region of the genomic sequence. In addition, the primersused in the PCR assay mixture can comprise at least one forward primersand at least one reverse primer. For example, the KASPar® PCR assaymixture can use two forward primers corresponding to two differentalleles and one reverse primer. One of the forward primers contains asequence corresponding to specific region of the endogenous genomicsequence. The second forward primer contains a sequence corresponding toa specific region of the DNA polynucleotide. The reverse primer containsa sequence corresponding to a specific region of the genomic sequence.Such a KASPar® assay for detection of an amplification reaction is anembodiment of the subject disclosure.

In some embodiments the fluorescent signal or fluorescent dye isselected from the group consisting of a HEX fluorescent dye, a FAMfluorescent dye, a JOE fluorescent dye, a TET fluorescent dye, a Cy 3fluorescent dye, a Cy 3.5 fluorescent dye, a Cy 5 fluorescent dye, a Cy5.5 fluorescent dye, a Cy 7 fluorescent dye, and a ROX fluorescent dye.

In other embodiments the amplification reaction is run using suitablesecond fluorescent DNA dyes that are capable of staining cellular DNA ata concentration range detectable by flow cytometry, and have afluorescent emission spectrum which is detectable by a real timethermocycler. It should be appreciated by those of ordinary skill in theart that other nucleic acid dyes are known and are continually beingidentified. Any suitable nucleic acid dye with appropriate excitationand emission spectra can be employed, such as YO-PRO-1®, SYTOX Green®,SYBR Green I®, SYTO11®, SYTO12®, SYTO13®, BOBO®, YOYO®, and TOTO®. Inone embodiment, a second fluorescent DNA dye is SYTO13® used at lessthan 10 μM, less than 4 μM, or less than 2.7 μM.

In further embodiments, Next Generation Sequencing (NGS) can be used fordetection. As described by Brautigma et al., 2010, DNA sequence analysiscan be used to determine the nucleotide sequence of the isolated andamplified fragment. The amplified fragments can be isolated andsub-cloned into a vector and sequenced using chain-terminator method(also referred to as Sanger sequencing) or Dye-terminator sequencing. Inaddition, the amplicon can be sequenced with Next Generation Sequencing.NGS technologies do not require the sub-cloning step, and multiplesequencing reads can be completed in a single reaction. Three NGSplatforms are commercially available, the Genome Sequencer FLX™ from 454Life Sciences/Roche, the Illumina Genome Analyser™ from Solexa andApplied Biosystems' SOLiD™ (acronym for: ‘Sequencing by Oligo Ligationand Detection’). In addition, there are two single molecule sequencingmethods that are currently being developed. These include the trueSingle Molecule Sequencing (tSMS) from Helicos Bioscience™ and theSingle Molecule Real Time™ sequencing (SMRT) from Pacific Biosciences.

The Genome Sequencher FLX™ which is marketed by 454 Life Sciences/Rocheis a long read NGS, which uses emulsion PCR and pyrosequencing togenerate sequencing reads. DNA fragments of 300-800 bp or librariescontaining fragments of 3-20 kb can be used. The reactions can produceover a million reads of about 250 to 400 bases per run for a total yieldof 250 to 400 megabases. This technology produces the longest reads butthe total sequence output per run is low compared to other NGStechnologies.

The Illumina Genome Analyser™ which is marketed by Solexa™ is a shortread NGS which uses sequencing by synthesis approach with fluorescentdye-labeled reversible terminator nucleotides and is based onsolid-phase bridge PCR. Construction of paired end sequencing librariescontaining DNA fragments of up to 10 kb can be used. The reactionsproduce over 100 million short reads that are 35-76 bases in length.This data can produce from 3-6 gigabases per run.

The Sequencing by Oligo Ligation and Detection (SOLiD) system marketedby Applied Biosystems™ is a short read technology. This NGS technologyuses fragmented double stranded DNA that are up to 10 kb in length. Thesystem uses sequencing by ligation of dye-labelled oligonucleotideprimers and emulsion PCR to generate one billion short reads that resultin a total sequence output of up to 30 gigabases per run.

tSMS of Helicos Bioscience™ and SMRT of Pacific Biosciences™ apply adifferent approach which uses single DNA molecules for the sequencereactions. The tSMS Helicos™ system produces up to 800 million shortreads that result in 21 gigabases per run. These reactions are completedusing fluorescent dye-labelled virtual terminator nucleotides that isdescribed as a ‘sequencing by synthesis’ approach.

The SMRT Next Generation Sequencing system marketed by PacificBiosciences™ uses a real time sequencing by synthesis. This technologycan produce reads of up to 1,000 bp in length as a result of not beinglimited by reversible terminators. Raw read throughput that isequivalent to one-fold coverage of a diploid human genome can beproduced per day using this technology.

In another embodiment, the detection can be completed using blottingassays, including Western blots, Northern blots, and Southern blots.Such blotting assays are commonly used techniques in biological researchfor the identification and quantification of biological samples. Theseassays include first separating the sample components in gels byelectrophoresis, followed by transfer of the electrophoreticallyseparated components from the gels to transfer membranes that are madeof materials such as nitrocellulose, polyvinylidene fluoride (PVDF), ornylon. Analytes can also be directly spotted on these supports ordirected to specific regions on the supports by applying vacuum,capillary action, or pressure, without prior separation. The transfermembranes are then commonly subjected to a post-transfer treatment toenhance the ability of the analytes to be distinguished from each otherand detected, either visually or by automated readers.

In a further embodiment the detection can be completed using an ELISAassay, which uses a solid-phase enzyme immunoassay to detect thepresence of a substance, usually an antigen, in a liquid sample or wetsample. Antigens from the sample are attached to a surface of a plate.Then, a further specific antibody is applied over the surface so it canbind to the antigen. This antibody is linked to an enzyme, and, in thefinal step, a substance containing the enzyme's substrate is added. Thesubsequent reaction produces a detectable signal, most commonly a colorchange in the substrate.

Transgenic Plants

In an embodiment, a plant, plant tissue, or plant cell comprises aBrachypodium distachyon metallothionein-like gene (mtl) promoter. In oneembodiment a plant, plant tissue, or plant cell comprises theBrachypodium distachyon metallothionein-like gene (mtl) promoter of asequence selected from SEQ ID NO:1 or a sequence that has at least 80%,85%, 90%, 95% or 99.5% sequence identity with a sequence selected fromSEQ ID NO:1. In one embodiment a plant, plant tissue, or plant cellcomprises the Brachypodium distachyon metallothionein-like gene (mtl)promoter of a sequence selected from SEQ ID NO:1 or a sequence that has80%, 85%, 90%, 95% or 99.5% sequence identity with a sequence selectedfrom SEQ ID NO:1. In another embodiment a plant, plant tissue, or plantcell comprises the Brachypodium distachyon metallothionein-like gene(mtl) 3′-UTR consists of a sequence selected from SEQ ID NO:3 or asequence that has at least 80%, 85%, 90%, 95% or 99.5% sequence identitywith a sequence selected from SEQ ID NO:3. In another embodiment aplant, plant tissue, or plant cell comprises the Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR consists of a sequence selectedfrom SEQ ID NO:3 or a sequence that has 80%, 85%, 90%, 95% or 99.5%sequence identity with a sequence selected from SEQ ID NO:3. In anembodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a sequence selected from SEQ ID NO:1, ora sequence that has at least 80%, 85%, 90%, 95% or 99.5% sequenceidentity with a sequence selected from SEQ ID NO:1 that is operablylinked to a non-Brachypodium distachyon metallothionein-like transgene.In an embodiment, a plant, plant tissue, or plant cell comprises a geneexpression cassette comprising a sequence selected from SEQ ID NO:1, ora sequence that has 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:1 that is operably linked to anon-Brachypodium distachyon metallothionein-like transgene. In anillustrative embodiment, a plant, plant tissue, or plant cell comprisesa gene expression cassette comprising a Brachypodium distachyonmetallothionein-like promoter that is operably linked to a transgene,wherein the transgene can be an insecticidal resistance transgene, anherbicide tolerance transgene, a nitrogen use efficiency transgene, awater use efficiency transgene, a nutritional quality transgene, a DNAbinding transgene, a selectable marker transgene, or combinationsthereof.

In accordance with one embodiment a plant, plant tissue, or plant cellis provided wherein the plant, plant tissue, or plant cell comprises anon-endogenous Brachypodium distachyon metallothionein-like gene (mtl)derived promoter sequence operably linked to a transgene, wherein theBrachypodium distachyon metallothionein-like gene (mtl) promoter derivedpromoter sequence comprises a sequence SEQ ID NO:1 or a sequence having80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1. In oneembodiment a plant, plant tissue, or plant cell is provided wherein theplant, plant tissue, or plant cell comprises SEQ ID NO: 1, or a sequencethat has at least 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQID NO: 1 operably linked to a non-Brachypodium distachyonmetallothionein-like transgene. In one embodiment a plant, plant tissue,or plant cell is provided wherein the plant, plant tissue, or plant cellcomprises SEQ ID NO: 1, or a sequence that has 80%, 85%, 90%, 95% or99.5% sequence identity with SEQ ID NO: 1 operably linked to anon-Brachypodium distachyon metallothionein-like transgene. In oneembodiment the plant, plant tissue, or plant cell is a dicotyledonous ormonocotyledonous plant or a cell or tissue derived from a dicotyledonousor monocotyledonous plant. In one embodiment the plant is selected fromthe group consisting of maize, wheat, rice, sorghum, oats, rye, bananas,sugar cane, soybean, cotton, sunflower, and canola. In one embodimentthe plant is Zea mays. In accordance with one embodiment the plant,plant tissue, or plant cell comprises SEQ ID NO: 1 or a sequence having80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1 operablylinked to a non-Brachypodium distachyon metallothionein-like transgene.In one embodiment the plant is Zea mays. In accordance with oneembodiment the plant, plant tissue, or plant cell comprises SEQ ID NO: 1or a sequence having at least 80%, 85%, 90%, 95% or 99.5% sequenceidentity with SEQ ID NO:1 operably linked to a non-Brachypodiumdistachyon metallothionein-like transgene. In one embodiment the plant,plant tissue, or plant cell comprises a promoter operably linked to atransgene wherein the promoter consists of SEQ ID NO: 1 or a sequencehaving 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1.In one embodiment the plant, plant tissue, or plant cell comprises apromoter operably linked to a transgene wherein the promoter consists ofSEQ ID NO: 1 or a sequence having at least 80%, 85%, 90%, 95% or 99.5%sequence identity with SEQ ID NO:1. In accordance with one embodimentthe gene construct comprising Brachypodium distachyonmetallothionein-like derived promoter sequence operably linked to atransgene is incorporated into the genome of the plant, plant tissue, orplant cell.

In one embodiment a non-Brachypodium plant, plant tissue, or plant cellis provided comprising SEQ ID NO: 1, or a sequence that has at least80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1, operablylinked to a transgene. In one embodiment a non-Brachypodium plant, planttissue, or plant cell is provided comprising SEQ ID NO: 1, or a sequencethat has 80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1,operably linked to a transgene. In accordance with one embodiment thenon-Brachypodium plant, plant tissue, or plant cell is a dicotyledonousor monocotyledonous plant or plant cell or tissue derived from adicotyledonous or monocotyledonous plant. In one embodiment the plant isselected from the group consisting of maize, wheat, rice, sorghum, oats,rye, bananas, sugar cane, soybean, cotton, sunflower, and canola. In oneembodiment the plant is Zea mays. In accordance with one embodiment thepromoter sequence operably linked to a transgene is incorporated intothe genome of the plant, plant tissue, or plant cell.

In one embodiment a non-Brachypodium plant, plant tissue, or plant cellis provided that comprises SEQ ID NO: 1, or a sequence that has at least80%, 85%, 90%, 95% or 99.5% sequence identity with SEQ ID NO:1, operablylinked to the 5′ end of a transgene and a 3′ untranslated sequencecomprising SEQ ID NO: 3 or a sequence that has at least 80%, 85%, 90%,95% or 99.5% sequence identity with SEQ ID NO:3, wherein the 3′untranslated sequence is operably linked to said transgene. Inaccordance with one embodiment the non-Brachypodium plant, plant tissue,or plant cell is a dicotyledonous or monocotyledonous plant or is aplant issue or cell derived from a dicotyledonous or monocotyledonousplant. In one embodiment the plant is selected from the group consistingof maize, wheat, rice, sorghum, oats, rye, bananas, sugar cane, soybean,cotton, sunflower, and canola. In one embodiment the plant is Zea mays.In accordance with one embodiment the promoter sequence operably linkedto a transgene is incorporated into the genome of the plant, planttissue, or plant cell.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a monocotyledonous plant. Themonocotyledonous plant, plant tissue, or plant cell can be, but notlimited to corn, rice, wheat, sugarcane, barley, rye, sorghum, orchids,bamboo, banana, cattails, lilies, oat, onion, millet, switchgrass,turfgrass, and triticale.

In an embodiment, a plant, plant tissue, or plant cell according to themethods disclosed herein can be a dicotyledonous plant. Thedicotyledonous plant, plant tissue, or plant cell can be, but notlimited to alfalfa, rapeseed, canola, Indian mustard, Ethiopian mustard,soybean, sunflower, cotton, beans, broccoli, cabbage, cauliflower,celery, cucumber, eggplant, lettuce; melon, pea, pepper, peanut, potato,pumpkin, radish, spinach, sugarbeet, sunflower, tobacco, tomato, andwatermelon.

One of skill in the art will recognize that after the exogenous sequenceis stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above, wherein the seed has the transgene or gene constructcontaining the gene regulatory elements of the subject disclosure. Thepresent disclosure further encompasses the progeny, clones, cell linesor cells of the transgenic plants described above wherein said progeny,clone, cell line or cell has the transgene or gene construct containingthe gene regulatory elements of the subject disclosure.

The present disclosure also encompasses the cultivation of transgenicplants described above, wherein the transgenic plant has the transgeneor gene construct containing the gene regulatory elements of the subjectdisclosure. Accordingly, such transgenic plants may be engineered to,inter alia, have one or more desired traits or transgenic eventscontaining the gene regulatory elements of the subject disclosure, bybeing transformed with nucleic acid molecules according to theinvention, and may be cropped or cultivated by any method known to thoseof skill in the art.

Method of Expressing a Transgene

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a Brachypodium distachyonmetallothionein-like gene (mtl) promoter operably linked to at least onetransgene or a polylinker sequence. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter 3′-UTR operably linked to at least one transgene or apolylinker sequence. In one embodiment the Brachypodium distachyonmetallothionein-like gene (mtl) promoter consists of a sequence selectedfrom SEQ ID NO:1 or a sequence that has at least 80%, 85%, 90%, 95% or99.5% sequence identity with a sequence selected from SEQ ID NO:1. Inanother embodiment the Brachypodium distachyon metallothionein-like gene(mtl) 3′-UTR consists of a sequence selected from SEQ ID NO:3 or asequence that has at least 80%, 85%, 90%, 95% or 99.5% sequence identitywith a sequence selected from SEQ ID NO:3. In an embodiment, a method ofexpressing at least one transgene in a plant comprising growing a plantcomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter and a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprising culturing a plant tissue or plant cell comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoteroperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant tissue or plant cellcomprising culturing a plant tissue or plant cell comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoter 3′-UTRoperably linked to at least one transgene. In an embodiment, a method ofexpressing at least one transgene in a plant tissue or plant cellcomprising culturing a plant tissue or plant cell comprising aBrachypodium distachyon metallothionein-like gene (mtl) promoter and aBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR operablylinked to at least one transgene.

In an embodiment, a method of expressing at least one transgene in aplant comprises growing a plant comprising a gene expression cassettecomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter operably linked to at least one transgene. In one embodimentthe Brachypodium distachyon metallothionein-like gene (mtl) promoterconsists of a sequence selected from SEQ ID NO:1 or a sequence that hasat least 80%, 85%, 90%, 95% or 99.5% sequence identity with a sequenceselected from SEQ ID NO:1. In one embodiment the Brachypodium distachyonmetallothionein-like gene (mtl) promoter consists of a sequence selectedfrom SEQ ID NO:1 or a sequence that has 80%, 85%, 90%, 95% or 99.5%sequence identity with a sequence selected from SEQ ID NO:1. In anotherembodiment the Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR consists of a sequence selected from SEQ ID NO:3 or a sequencethat has at least 80%, 85%, 90%, 95% or 99.5% sequence identity with asequence selected from SEQ ID NO:3. In another embodiment theBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR consistsof a sequence selected from SEQ ID NO:3 or a sequence that has 80%, 85%,90%, 95% or 99.5% sequence identity with a sequence selected from SEQ IDNO:3. In an embodiment, a method of expressing at least one transgene ina plant comprises growing a plant comprising a gene expression cassettecomprising a Brachypodium distachyon metallothionein-like gene (mtl)promoter and a Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant comprises growinga plant comprising a gene expression cassette comprising a Brachypodiumdistachyon metallothionein-like gene (mtl) 3′-UTR operably linked to atleast one transgene. In an embodiment, a method of expressing at leastone transgene in a plant tissue or plant cell comprises culturing aplant tissue or plant cell comprising a gene expression cassettecontaining a Brachypodium distachyon metallothionein-like gene (mtl)promoter operably linked to at least one transgene. In an embodiment, amethod of expressing at least one transgene in a plant tissue or plantcell comprises culturing a plant tissue or plant cell comprising a geneexpression cassette containing a Brachypodium distachyonmetallothionein-like gene (mtl) 3′-UTR operably linked to at least onetransgene. In an embodiment, a method of expressing at least onetransgene in a plant tissue or plant cell comprises culturing a planttissue or plant cell comprising a gene expression cassette, aBrachypodium distachyon metallothionein-like gene (mtl) promoter and aBrachypodium distachyon metallothionein-like gene (mtl) 3′-UTR operablylinked to at least one transgene.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. The examples should not be construed tolimit the disclosure to the particular features or embodimentsexemplified.

EXAMPLES Example 1 A Promoter from a Brachypodium distachyonMetallothionein-like Gene (Mtl)

The promoter from a Brachypodium distachyon metallothionein-like gene(mtl) (SEQ ID NO:1) is a 2,000 bp polynucleotide sequence that wasidentified from the Brachypodium distachyon genomic DNA (gDNA) sequence.The promoter sequence was identified by BLASTing the Phytozome database(Goodstein et al., 2012) with a Zea mays metallothionein-like gene(mtl). The resulting hits were analyzed and a single coding sequence wasselected for further analysis. From the assessment of the contiguouschromosomal sequence that spanned millions of base pairs, a 2,000 bppolynucleotide sequence was identified and isolated for use inexpression of heterologous coding sequences. This novel polynucleotidesequence was analyzed for use as a regulatory sequence to driveexpression of a gene. As shown in the sequence (SEQ ID NO:2) below, the2,000 bp Brachypodium distachyon metallothionein-like gene (mtl)promoter of SEQ ID NO:1 is provided as base pairs 1-2,000. The nativemtl gene coding sequence of SEQ ID NO:6 is provided as base pairs2,001-2,345. The 1,002 bp Brachypodium distachyon metallothionein-likegene (mtl) 3′-UTR of SEQ ID NO:3 is provided as base pairs 2,346-3,347.Accordingly, SEQ ID NO:2 is provided as:

tttcgtgtgcttacttggggcttcctttgggcctgcggaagcagacggcgtgccagctgagaccgttggtggacaatgtggatggacgtctagcttcgtggaaggcccttctcctttccaagggaggatgactggtgctggtcaagtccaccctgacagcgatgccggtctactcgatgatgtcactcgacctttcggacaagatcatcaaaggggtggacaagatctgtcgtggtttcctttggcgtggtcagaaggacgctaaaggggggggggcattgtatggtggcttgggtcgtggtgtgttcgccgaagcctttcggtggactcggaattccaaatcttcgcatgctcaacgaggccttgagggtccgctagaggtggcttgagagggtggacgattccaaaccatgggtcggtttcaaggttgagacgcccagaggtctatgttatctttgaggcggccacttcctctttggtgggcgatggccggaagaccctcttctggaccgatcgttggctgcatggcgtctgttttcgagatgcgttcctgatcctcgtgggccatgtttgccacaagtttttgcgcacgcgcacagtgcgggaggctctgcatggagcgtggactggcgacatgggtccagacctaaagggaagctactcttgaggagttcctcactctctgggactggcttcgtggggtccaactggaagaagggagggccgactctcttcattggaactggtctcgtggtgatagttactcggcaaagactgcttatgcgaatctcttcactggccgcatggttgacccgttagcggctgagatctagggcttcggagaggtgtcgtttcttcgtctggctcgcagttcgaaatagatgctagacggcggatcgcttgcgaggcgtgggcttccccaccctgacaagtgtcaggagatggagaccatttcccatctactcttggggtgtgtcttggcgaggcaggtttgggagcgcttgtgggcggcttggggacacgtggagtgggctccgaatgcggatgctactctgcgggaatggtggtcttctcttcccttaccacggcgagcccggagggacttccggacggggatcatacttgttctttggaccatttggtgccattggaatgatgtggccttccttgcagcttgtcgtgcttcgcctccaggaggagtttggtcggtgggcgcacgccggcctctttaggggtagagtgtctattccagccttgatggtgagtggttggatagctagcacatagtctttttttctctttcttgccacttggtgatgttgtattgccgcttcggcgttgtactagccttctggctcttcttatttaatcgatgatgcacccgctgggtggcattcttgaaatttttttgagaaaatactcctacaaaggaagtacagtaatttgatacacaatgctttctctaaatacgaaaatacaaacatcctaatgttattccaactatgctactccctccgttcctaaactcttatctttgtttttgttcaaatttgtaccaaacaacgacaaaaatttagtaacggaggaagtatatatacgaagtgataccaaataaggtgtttctttacttgaaagctagttcctctgaggtaaaagaaatgatgaggatcaaggaaggcccatttcttgccggcacgcagttgccgaagtcagtagatttaaaatgacctcacatactgaagaaggaaagggaagttaaccacgccattgttgctttagcagaaagcaaccatatcctaattaacatcacagtacacgttgaaacggcagggcacgataattggtgaagactgattccagggagcatcgtggcacgcaaaaacgcccttgccttgttcccttataaatagtggtgcagaagatacatttgtgatcaccatctcaaagcgcctctttattcccctttcatcttgagcttaagtactagagaaaaattaaggatgtcttgcagctgtggatctggatgcagctgcggctcaaactgcacctgcgggtatgtatagatgcttaattctgtgttgcagattactacgtagctgctaataccatctaagtaatgacaatgctttctcatgatgatcaattttcatttgctgtgtggttcagcaagatgtaccctgacttggcagagaagagcggcggcacccagcaggccaccaccatgatcctcggcgttgcgcctgctaaggcccagtttgaggaggccgccgagtccggtgaggccggccatggctgcagctgcggcgccaactgcaagtgcgacccgtgcaactgctaagatgcacgcgacgcgctgctgcttgtggtgtgtgtttgtgtgatctcgactgaataaaagggacacggtacatccggggttggttggtgctcgagtcgagtgtgcaaggttgtgtggtttgccagctcttggtgtgtgtctccttgtgctcatgtgacttgtgaaaccattaatagtaaccactttgtgcttgtgtgtgtgtgggcacctgtgtttctgtaatggtctatctggagtgaatatatacaagacaggtttgcctaaccatgccttttccttccttgaggtcatcgcgccaacacgaatgacgagaaccaatgatcttaggactatcaaatagacaatataactcagcatggagcaatgtactgctaatgaacggtcctaatcaatacagttgggaagcagtaatcgatggaaaacacttcctcagttcagaagggaacatgtagatgggaaagtaaacaggatatatagacacatgcagttaggtctaccacaagaagacatgccacaggactaaaaacagaagctgggtttggaacaacttatgcagacgctagctatccagataaaccttagagaaacagctatgagaataagcgtctgaaatttgtagttgcctgtttggaatggcttatggacgacaagcttattttctggagtagcctacaagtacaagccaaggcacatgctgttccaaactggcccatattttgcacaaccaaaggaaaagagacagtgacataagagcatctccagtgattcagtgagataccccaaagtcatccaaatgtccgtttcggccgaaatggacatgctggccagaaaaacatctcccaatgggttaccccaaatagatattttgtatgttttgtccggcccaatccccaaacatctcctaaatttggggcaactttgcggtgtccagtgtccggtgtccgggcccacttctttttctcccaagcgagcatcttcctcccgaagcacgaagcccccgtc

Example 2 Vector Construction

The following vectors were built to incorporate the promoter from aBrachypodium distachyon metallothionein-like gene (mtl) upstream of atransgene. The vector construct pDAB120419 contained a gene expressioncassette, in which the phi-yfp transgene (Phi-yellow fluorescentprotein; Clontech, Mountain View, Calif.) was driven by the promoterfrom a Brachypodium distachyon metallothionein-like gene (mtl) of SEQ IDNO:1 and flanked by the Brachypodium distachyon metallothionein-likegene (mtl) 3′-UTR of SEQ ID NO:3. A diagram of this gene expressioncassette is shown in FIG. 1 and is provided as SEQ ID NO:4. The vectoralso contained a selectable marker gene expression cassette thatcontained the aad-1 transgene (U.S. Pat. No. 7,838,733) driven by theZea mays Ubiquitin 1 promoter (Christensen et al., (1992) PlantMolecular Biology 18; 675-689) and was terminated by the Zea mays Lipase3′-UTR (U.S. Pat. No. 7,179,902). A diagram of this gene expressioncassette is shown in FIG. 1 and is provided as SEQ ID NO:5. Thisconstruct was built by synthesizing the newly designed promoter from aBrachypodium distachyon metallothionein-like gene by an externalprovider (Geneart via Life Technologies, Carlsbad, Calif.), and cloningthe promoter into a Gateway™ (Life Technologies) donor vector using theGeneArt® Seamless Cloning and Assembly Kit (Life technologies) andrestriction enzymes. The resulting donor vector was integrated into afinal binary destination vector using the Gateway™ cloning system (LifeTechnologies). Clones of pDAB120419 were obtained and confirmed viarestriction enzyme digestions and sequencing. The resulting constructcontained a promoter that could robustly drive expression of atransgenes which was operably linked to the 3′ end of the promoter.

Example 3 Modified Brachypodium distachyon Metallothionein-like Gene(Mtl) Regulatory Elements

The 2,000 bp Brachypodium distachyon metallothionein-like gene (mtl)promoter of SEQ ID NO:1 was modified by making alterations to thepromoter sequence. A variant was designed and provided as SEQ ID NO:12.An alignment comparing the modified Brachypodium distachyonmetallothionein-like gene (mtl) promoter of SEQ ID NO:1 and the modifiedBrachypodium distachyon metallothionein-like gene (mtl) promoter of SEQID NO:12 is provided in FIG. 3. As shown in FIG. 3 the promoterpolynucleotide sequences of SEQ ID NO:1 and SEQ ID NO:12 share 97.2%sequence identity. Herein are provide novel promoter sequences thatoriginated from the Brachypodium distachyon metallothionein-like gene(mtl) promoters of SEQ ID NO:1. In an embodiment, the novel promoterpolynucleotide sequence of SEQ ID NO:12 can be used in a gene expressioncassette to drive the expression of transgenes. In another aspect ofthis embodiment, the novel promoter polynucleotide sequence of SEQ IDNO:12 can be operably linked to a gene of interest and flanked by a3′-UTR, for example the 3′-UTR of SEQ ID NO:3, and can be used in a geneexpression cassette to drive the expression of transgenes.

The 1,002 bp Brachypodium distachyon metallothionein-like gene (mtl)3′-UTR of SEQ ID NO:3 was modified by making alterations to the 3′-UTRsequence. New variants were designed and are provided as SEQ ID NO:17and SEQ ID NO:18. An alignment comparing the modified Brachypodiumdistachyon metallothionein-like gene (mtl) 3′-UTR of SEQ ID NO:3 and themodified Brachypodium distachyon metallothionein-like gene (mtl) 3′-UTRsof SEQ ID NO:17 and SEQ ID NO:18 is provided in FIG. 5. As shown in FIG.5 the 3′-UTR polynucleotide sequences of SEQ ID NO:3 and SEQ ID NO:17share 94.2% sequence identity. Depicted in FIG. 5 are the 3′-UTRpolynucleotide sequences of SEQ ID NO:3 and SEQ ID NO:18 which share97.5% sequence identity. Finally, FIG. 5 presents the 3′-UTRpolynucleotide sequences of SEQ ID NO:18 and SEQ ID NO:17 which share92.7% sequence identity. Herein is provided a novel 3′-UTR sequence thatoriginated from the Brachypodium distachyon metallothionein-like gene(mtl) 3′-UTR of SEQ ID NO:3. In an embodiment, the novel 3′-UTRpolynucleotide sequence of SEQ ID NO:17 can be used in a gene expressioncassette to terminate the expression of transgenes. In another aspect ofthis embodiment, the novel 3′-UTR polynucleotide sequence of SEQ IDNO:17 can be operably linked to a transgene driven by a promoter, forexample the promoter of SEQ ID NO:1, and can be used in a geneexpression cassette to drive the expression of transgenes. In a furtherembodiment, the novel 3′-UTR polynucleotide sequence of SEQ ID NO:18 canbe used in a gene expression cassette to terminate the expression oftransgenes. In another aspect of this embodiment, the novel 3′-UTRpolynucleotide sequence of SEQ ID NO:18 can be operably linked to atransgene driven by a promoter, for example the promoter of SEQ ID NO:1,and can be used in a gene expression cassette to drive the expression oftransgenes.

Example 4 Maize Transformation

Transformation of Agrobacterium tumefaciens:

The binary expression vector was transformed into Agrobacteriumtumefaciens strain DAt13192 (RecA deficient ternary strain) (Int'l. Pat.Pub. No. WO2012016222). Bacterial colonies were selected, and binaryplasmid DNA was isolated and confirmed via restriction enzyme digestion.

Agrobacterium Culture Initiation:

Agrobacterium cultures were streaked from glycerol stocks onto ABminimal medium (Gelvin, S., 2006, Agrobacterium Virulence GeneInduction, in Wang, K., ed., Agrobacterium Protocols Second Edition Vol.1, Humana Press, p. 79; made without sucrose and with 5 g/L glucose and15 g/L Bacto™ Agar) and incubated at 20° C. in the dark for 3 days.Agrobacterium cultures were then streaked onto a plate of YEP medium(Gelvin, S., 2006, Agrobacterium Virulence Gene Induction, in Wang, K.,ed., Agrobacterium Protocols Second Edition Vol. 1, Humana Press, p. 79)and incubated at 20° C. in the dark for 1 day.

On the day of an experiment, a mixture of Inoculation medium (2.2 g/L MSsalts, 68.4 g/L sucrose, 36 g/L glucose, 115 mg/L L-proline, 2 mg/Lglycine, 100 mg/L myo-Inositol, 0.05 mg/L nicotinic acid, 0.5 mg/Lpyridoxine HCl, 0.5 mg/L thiamine HCl) and acetosyringone was preparedin a volume appropriate to the size of the experiment. A 1 M stocksolution of acetosyringone in 100% dimethyl sulfoxide was added to theInoculation medium to make a final acetosyringone concentration of 200μM.

For each construct, 1-2 loops of Agrobacterium from the YEP plate weresuspended in 15 ml of the inoculation medium/acetosyringone mixtureinside a sterile, disposable, 50 ml centrifuge tube and the opticaldensity of the solution at 600 nm (O.D.₆₀₀) was measured in aspectrophotometer. The suspension was then diluted down to 0.25-0.35O.D.₆₀₀ using additional Inoculation medium/acetosyringone mixture. Thetube of Agrobacterium suspension was then placed horizontally on aplatform shaker set at about 75 rpm at room temperature for between 1and 4 hours before use.

Maize Transformation:

Experimental constructs were transformed into maize viaAgrobacterium-mediated transformation of immature embryos isolated fromthe inbred line, Zea mays c.v. B104. The method used is similar to thosepublished by Ishida et al., (1996) Nature Biotechnol 14:745-750 andFrame et al., (2006) Plant Cell Rep 25: 1024-1034, but with severalmodifications and improvements to make the method amenable tohigh-throughput transformation. An example of a method used to produce anumber of transgenic events in maize is given in U.S. Pat. App. Pub. No.US 2013/0157369 A1, beginning with the embryo infection andco-cultivation steps.

Putative T₀ transgenic plantlets were transplanted from Phytatrays™(Sigma-Aldrich; St. Louis, Mo.) to small 3.5″ plastic pots (T. O.Plastics; Clearwater, Minn.) filled with growing media (Premix BX;Premier Tech Horticulture), covered with humidomes (Arco Plastics Ltd.),and then hardened-off in a growth room (28° C. day/24° C. night, 16-hourphotoperiod, 50-70% RH, 200 μEm-2 sec-1 light intensity). When plantsreached the V3-V4 developmental stage (3-4 leaf collars visible), theywere transplanted into Sunshine Custom Blend 160 soil mixture and grownto flowering in the greenhouse (Light Exposure Type: Photo orAssimilation; High Light Limit: 1200 PAR; 16-hour day length; 27° C.day/24° C. night). The plants were analyzed for transgene copy number byqPCR assays using primers designed to detect relative copy numbers ofthe transgenes, and putative single copy events selected for advancementwere transplanted into 5 gallon pots.

Example 5 Molecular Confirmation of Copy Number

The stable integration of the phi-yfp transgene within the genome of thetransgenic Z. mays plants was confirmed via a hydrolysis probe assay.Stably-transformed transgenic Z. mays plantlets that developed from thecallus were obtained and analyzed to identify events that contained alow copy number (1-2 copies) of full-length T-strand inserts. Identifiedplantlets were advanced to the green house and grown.

The Roche Light Cycler480™ system was used to determine the transgenecopy number. The method utilized a biplex TaqMan® reaction that employedoligonucleotides specific to the phi-yfp gene and to the endogenous Z.mays reference gene, invertase (Genbank Accession No: U16123.1), in asingle assay. Copy number and zygosity were determined by measuring theintensity of phi-yfp specific fluorescence, relative to theinvertase-specific fluorescence, as compared to known copy numberstandards.

A phi-yfp gene-specific DNA fragment was amplified with one TaqMan®primer/probe set containing a probe labeled with FAM™ fluorescent dye,and invertase was amplified with a second TaqMan® primer/probe setcontaining a probe labeled with HEX™ fluorescence. Copy number andzygosity of the samples were determined by measuring the relativeintensity of fluorescence specific for the reporter gene, phi-yfp, tofluorescence specific for the reference gene, invertase, as compared toknown copy number standards.

Example 6 Molecular Confirmation of Protein Expression

Protein Extraction:

Nunc® 96-well Maxi-Sorp Plates (Thermo Fisher Scientific Inc., Rockford,Ill.) were used for ELISA. The plates were coated with mouse monoclonalanti Phi-YFP capture antibody (OriGene Technologies Inc., Rockville,Md.). The antibody was diluted in PBS (1 μg/mL) and 150 μL of dilutedPBS was added per well. The plates were incubated overnight at 4° C. Theplates were kept at room temperature for 20-30 minutes before washing 4×with 350 μL of wash buffer [1×PBS supplemented with 0.05% Tween®-20(Sigma-Aldrich, St. Louis, Mo.)]. Next, the plates were blocked with 200μL per well of blocking buffer [1×PBS supplemented with 0.05% Tween®-20plus 0.5% BSA (Millipore Probumin®)] for a minimum of 1 hr at +37° C.followed by 4× washing with 350 μL of wash buffer (Tomtec QuadraWash™ 2,Tomtec, Inc., Hamden, Conn.).

For the Phi-YFP ELISA, Evrogen recombinant Phi-YFP 1 mg/mL (Axxora LLC,Farmingdale, N.Y.) was used as a standard. A 5-parameter fit standardcurve (between the 1 ng/ml and 0.125 ng/ml standards) was used to ensureall data fall in the linear portion of the curve. Next, 100 μL ofstandard or sample was added to the well. A minimum 1:4 dilution ofsample in the Assay Buffer was used. Plates were incubated for 1 hour atRT on plate shaker (250 rpm; Titer Plate shaker) followed by 4× washingwith 350 μL of wash buffer (Tomtec QuadraWash™ 2). About 100 μL of 1μg/mL Evrogen rabbit polyclonal anti Phi-YFP primary antibody (Axxora)was added to each well. Plates were incubated for 1 hour at roomtemperature on a plate shaker at 250 rpm followed by 4× washing with 350μL of wash buffer (Tomtec QuadraWash™ 2). Next, 100 μL of anti-rabbitIgG HRP secondary antibody (Thermo Scientific) diluted 1:5000 inBlocking/Assay buffer, which was added to each well. Plates wereincubated for 1 hour at room temperature on plate shaker at 250 rpmfollowed by 4× washes with 350 μL of wash buffer (Tomtec QuadraWash™ 2).Then 100 μL of Pierce 1 Step Ultra TMB ELISA™ (Thermo Scientific)substrate was added in the well with gentle shaking for 10 minutes. Thereaction was stopped by adding 50 μL of 0.4N H2504. Absorbance was readat 450 nm with a 650 nm reference filter.

The Phi-YFP expression levels were determined by ELISAs using kits fromAcadia BioSciences (Portland, Me.). The ELISAs were performed usingmultiple dilutions of the extracts and using the reagents andinstructions provided by the supplier. The protein levels werenormalized using total soluble protein assay, performed using the 660 nmprotein assay reagent supplied by Thermo Scientific and following thesupplier's instructions.

Example 7 Expression of Genes Operably Linked to the Promoter from aBrachypodium distachyon Metallothionein-like Gene (Mtl)

Maize plants were transformed with a gene expression construct thatcontained the promoter from a Brachypodium distachyonmetallothionein-like gene (mtl) as described above. The ELISA analysisconfirmed that the novel promoter drove robust expression of atransgene. The quantitative measurements of Phi-YFP protein obtainedfrom transgenic plants comprising novel promoter constructs are shown inTABLE 1. The data show that Phi-YFP protein in the plants containing thenovel Brachypodium distachyon metallothionein-like gene (mtl) promoter(i.e., pDAB120419) is expressed preferentially higher in root tissue ascompared to the leaf tissue.

TABLE 1 B. distachyon MTL Promoter T0 Expression of Phi-YFP and AAD1PhiYFP (ng/mg) AAD1 (ng/mg) Construct Event Leaf Root Leaf Root 120419120419[1]-002.001 3 ND 107 ND 120419 120419[1]-003.001 3 ND 116 ND120419 120419[1]-005.001 3 ND 392 ND 120419 120419[1]-007.001 3 22 149129 120419 120419[1]-010.001 3 ND 110 ND 120419 120419[1]-011.001 3 55111  62 120419 120419[1]-014.001 3 ND 162 ND 120419 120419[1]-017.001 3ND 324 ND 120419 120419[1]-018.001 3 607  412 372 120419120419[1]-023.001 3 ND 103 ND 120419 120419[1]-024.001 3 ND 107 ND120419 120419[1]-026.001 3 346  289 147 120419 120419[1]-027.001 3 234 415 177 120419 120419[1]-031.001 3 41 900 233 120419 120419[1]-032.001 3ND 175 ND 120419 120419[1]-033.001 3 ND 313 ND 120419 120419[1]-035.0013 79 651 210 ND—Not Determined

T₀ single transgene copy plants containing a stably integratedpDAB120419 transgenic event were backcrossed to wild type B104 cornplants to obtain T₁ seed that were grown to mature plants. From thesemature plants, hemizygous T₁ plants were used for analysis to determinethe expression of the AAD1 and Phi-YFP protein. Five events perconstruct and ten plants per event were used the protein expressionanalysis. Three events per construct and three to five plants per eventwere used for other tissue type expression. Zygosity analysis was donefor Phi-YFP and AAD1. The quantitative ELISA measurements of Phi-YFP andAAD1 protein obtained from different tissue types including leaf, cob,husk, pollen, root, silk and stem tissue of T₁ transgenic plantscomprising novel promoter constructs are shown in TABLE 2. The dataconfirmed the T₀ leaf expression results and further showed thatconsistent high expression of Phi-YFP protein was obtained in theprimarily in root tissue types of the plants containing the novelpromoter (pDAB120419). These data demonstrate that the novel promoterillustrated here drives root preferential expression of transgene inplants, and that this expression profile is heritable from a firstgeneration to a second generation. Accordingly, the promoter from aBrachypodium distachyon metallothionein-like gene (mtl) is useful forbiotechnological applications.

TABLE 2 Protein expression in different tissue types of transgenic cornplants Mean (Phi- Mean (AAD1 Construct Events Tissue YFP ng/mg) ng/mg)120419 120419[1]-002 Leaf V4 0.3 34.5 120419 120419[1]-002 Root V4 608.72419.7 120419 120419[1]-014 Leaf V4 0.0 32.4 120419 120419[1]-014 RootV4 418.0 2375.7 120419 120419[1]-023 Cob R3 0.0 2794.7 120419120419[1]-023 Husk R3 0.0 4495.0 120419 120419[1]-023 Leaf R3 47.41387.0 120419 120419[1]-023 Leaf V12 0.0 188.3 120419 120419[1]-023 LeafV4 0.1 30.3 120419 120419[1]-023 Pollen R3 0.0 1003.7 120419120419[1]-023 Root R3 0.0 355.0 120419 120419[1]-023 Root V4 274.31536.0 120419 120419[1]-023 Silk R3 0.7 4515.0 120419 120419[1]-023 StemR3 1.7 6545.7 120419 120419[1]-024 Cob R3 0.0 3615.7 120419120419[1]-024 Husk R3 0.0 4998.3 120419 120419[1]-024 Kernel R3 0.02100.0 120419 120419[1]-024 Leaf R3 3.2 1498.2 120419 120419[1]-024 LeafV12 0.0 240.3 120419 120419[1]-024 Leaf V4 0.9 38.8 120419 120419[1]-024Pollen R3 0.0 4313.0 120419 120419[1]-024 Root R3 0.0 964.7 120419120419[1]-024 Root V4 132.0 1001.0 120419 120419[1]-024 Silk R3 0.37784.3 120419 120419[1]-024 Stem R3 0.0 5991.0 120419 120419[1]-033 CobR3 0.0 4212.7 120419 120419[1]-033 Husk R3 0.0 5691.0 120419120419[1]-033 Kernel R3 0.0 1804.0 120419 120419[1]-033 Leaf R3 0.31463.5 120419 120419[1]-033 Leaf V12 0.0 398.4 120419 120419[1]-033 LeafV4 0.6 30.7 120419 120419[1]-033 Pollen R3 0.0 3759.3 120419120419[1]-033 Root R3 0.0 1461.0 120419 120419[1]-033 Root V4 787.33445.0 120419 120419[1]-033 Silk R3 0.0 7656.3 120419 120419[1]-033 StemR3 0.0 6670.0

The Phi-YFP ELISA results indicated that the Brachypodium distachyonmetallothionein-like gene (mtl) promoter regulatory element (SEQ IDNO:1) drove below-ground preferred expression, specifically in roottissues, of Phi-YFP in T₀ events that were transformed with construct,pDAB120419. Negligible expression of Phi-YFP by the Brachypodiumdistachyon metallothionein-like gene (mtl) promoter regulatory elementwas observed in the leaf tissues of these events (TABLE 1 and TABLE 2).The events produced from the transformation also expressed AAD1 proteinin both leaf and root tissues. The expression of AAD1 within theseevents served as a control to compare expression levels of Phi-YFP indiffering tissues. In summary, the Brachypodium distachyonmetallothionein-like gene (mtl) promoter was developed for robustexpression of a transgenes within below ground tissues in plant specieslike corn.

Example 8 Crop Transformation of Genes Operably Linked to the Promoterfrom a Brachypodium distachyon Metallothionein-like Gene (Mtl)

Soybean may be transformed with genes operably linked to the promoterfrom a Brachypodium distachyon metallothionein-like gene (mtl) byutilizing the same techniques previously described in Example #11 orExample #13 of patent application WO 2007/053482.

Cotton may be transformed with genes operably linked to the promoterfrom a Brachypodium distachyon metallothionein-like gene (mtl) byutilizing the same techniques previously described in Examples #14 ofU.S. Pat. No. 7,838,733 or Example #12 of patent application WO2007/053482 (Wright et al.).

Canola may be transformed with genes operably linked to the promoterfrom a Brachypodium distachyon metallothionein-like gene (mtl) byutilizing the same techniques previously described in Example #26 ofU.S. Pat. No. 7,838,733 or Example #22 of patent application WO2007/053482 (Wright et al.).

Wheat may be transformed with genes operably linked to the promoter froma Brachypodium distachyon metallothionein-like gene (mtl) by utilizingthe same techniques previously described in Example #23 of patentapplication WO 2013/116700A1 (Lira et al.).

Rice may be transformed with genes operably linked to the promoter froma Brachypodium distachyon metallothionein-like gene (mtl) by utilizingthe same techniques previously described in Example #19 of patentapplication WO 2013/116700A1 (Lira et al.).

Example 9 Agrobacterium-mediated Transformation of Genes Operably Linkedto the Promoter from a Brachypodium distachyon Metallothionein-like Gene(Mtl)

In light of the subject disclosure, additional crops can be transformedaccording to embodiments of the subject disclosure using techniques thatare known in the art. For Agrobacterium-mediated transformation of rye,see, e.g., Popelka J C, Xu J, Altpeter F., “Generation of rye with lowtransgene copy number after biolistic gene transfer and production of(Secale cereale L.) plants instantly marker-free transgenic rye,”Transgenic Res. 2003 October; 12(5):587-96.). For Agrobacterium-mediatedtransformation of sorghum, see, e.g., Zhao et al.,“Agrobacterium-mediated sorghum transformation,” Plant Mol Biol. 2000December; 44(6):789-98. For Agrobacterium-mediated transformation ofbarley, see, e.g., Tingay et al., “Agrobacterium tumefaciens-mediatedbarley transformation,” The Plant Journal, (1997) 11: 1369-1376. ForAgrobacterium-mediated transformation of wheat, see, e.g., Cheng et al.,“Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens,”Plant Physiol. 1997 November; 115(3):971-980. For Agrobacterium-mediatedtransformation of rice, see, e.g., Hiei et al., “Transformation of ricemediated by Agrobacterium tumefaciens,” Plant Mol. Biol. 1997 September;35(1-2):205-18.

The latin names for these and other plants are given below. It should beclear that other (non-Agrobacterium) transformation techniques can beused to transform genes operably linked to the promoter from aBrachypodium distachyon Metallothionein-like gene (mtl), for example,into these and other plants. Examples include, but are not limited to;Maize (Zea mays), Wheat (Triticum spp.), Rice (Oryza spp. and Zizaniaspp.), Barley (Hordeum spp.), Cotton (Abroma augusta and Gossypiumspp.), Soybean (Glycine max), Sugar and table beets (Beta spp.), Sugarcane (Arenga pinnata), Tomato (Lycopersicon esculentum and other spp.,Physalis ixocarpa, Solanum incanum and other spp., and Cyphomandrabetacea), Potato (Solanum tuberosum), Sweet potato (Ipomoea batatas),Rye (Secale spp.), Peppers (Capsicum annuum, chinense, and frutescens),Lettuce (Lactuca sativa, perennis, and pulchella), Cabbage (Brassicaspp.), Celery (Apium graveolens), Eggplant (Solanum melongena), Peanut(Arachis hypogea), Sorghum (Sorghum spp.), Alfalfa (Medicago sativa),Carrot (Daucus carota), Beans (Phaseolus spp. and other genera), Oats(Avena sativa and strigosa), Peas (Pisum, Vigna, and Tetragonolobusspp.), Sunflower (Helianthus annuus), Squash (Cucurbita spp.), Cucumber(Cucumis sativa), Tobacco (Nicotiana spp.), Arabidopsis (Arabidopsisthaliana), Turfgrass (Lolium, Agrostis, Poa, Cynodon, and other genera),Clover (Trifolium), Vetch (Vicia). Transformation of such plants, withgenes operably linked to the promoter from a Brachypodium distachyonMetallothionein-like gene (mtl), for example, is contemplated inembodiments of the subject disclosure.

Use of the promoter from a Brachypodium distachyon Metallothionein-likegene (mtl) to drive operably linked genes can be deployed in manydeciduous and evergreen timber species. Such applications are alsowithin the scope of embodiments of this disclosure. These speciesinclude, but are not limited to; alder (Alnus spp.), ash (Fraxinusspp.), aspen and poplar species (Populus spp.), beech (Fagus spp.),birch (Betula spp.), cherry (Prunus spp.), eucalyptus (Eucalyptus spp.),hickory (Carya spp.), maple (Acer spp.), oak (Quercus spp.), and pine(Pinus spp.).

Use of the promoter from a Brachypodium distachyon Metallothionein-likegene (mtl) to drive operably linked genes can be deployed in ornamentaland fruit-bearing species. Such applications are also within the scopeof embodiments of this disclosure. Examples include, but are not limitedto; rose (Rosa spp.), burning bush (Euonymus spp.), petunia (Petuniaspp.), begonia (Begonia spp.), rhododendron (Rhododendron spp.),crabapple or apple (Malus spp.), pear (Pyrus spp.), peach (Prunus spp.),and marigolds (Tagetes spp.).

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A gene expression cassette comprising a promoteroperably linked to a heterologous nucleic acid, wherein the promotercomprises a polynucleotide comprising a sequence identity of at least97% to SEQ ID NO:1, and said promoter has below ground tissue preferredexpression.
 2. The gene expression cassette of claim 1, wherein thepolynucleotide further comprises an intron.
 3. The gene expressioncassette of claim 1, wherein the polynucleotide further comprises a 5′UTR.
 4. The gene expression cassette of claim 1, wherein thepolynucleotide is operably linked to a 3′ UTR.
 5. The gene expressioncassette of claim 4, wherein the 3′ UTR comprises SEQ ID NO:3.
 6. Thegene expression cassette of claim 1, wherein the operably linkedheterologous nucleic acid encodes a polypeptide or a small RNA gene. 7.The gene expression cassette of claim 1, wherein the heterologousnucleic acid is selected from the group consisting of a heterologousnucleic acid conferring insecticidal resistance, a heterologous nucleicacid conferring herbicide tolerance, a heterologous nucleic acidconferring nitrogen use efficiency, a heterologous nucleic acidconferring water use efficiency, a heterologous nucleic acid conferringnutritional quality, a heterologous nucleic acid encoding a DNA bindingprotein, and a heterologous nucleic acid encoding a selectable marker.8. A recombinant vector comprising the gene expression cassette of claim1, wherein the vector is selected from the group consisting of aplasmid, a cosmid, a bacterial artificial chromosome, a virus, and abacteriophage.
 9. A transgenic cell comprising the gene expressioncassette of claim
 1. 10. The transgenic cell of claim 9, wherein thetransgenic cell is a transgenic plant cell.
 11. A transgenic plantcomprising the transgenic plant cell of claim
 10. 12. The transgenicplant of claim 11, wherein the transgenic plant is a monocotyledonousplant or a dicotyledonous plant.
 13. The transgenic plant of claim 12,wherein the monocotyledonous plant is selected from the group consistingof a maize plant, a rice plant, and a wheat plant.
 14. A transgenic seedfrom the transgenic plant of claim 12, wherein the seed comprises thegene expression cassette.
 15. The gene expression cassette of claim 1,wherein the promoter comprises the polynucleotide sequence ofnucleotides 1 to 2,000 of SEQ ID NO:1.
 16. A method for expressing acoding sequence in a transgenic plant, the method comprising: a)transforming a plant cell with a gene expression cassette comprising apolynucleotide sequence comprising a sequence identity of at least 97%to SEQ ID NO:1 operably linked to a heterologous coding sequence, whichis operably linked to a 3′ untranslated region; b) isolating thetransformed plant cell comprising the gene expression cassette; c)regenerating a transgenic plant from the transformed plant cell; and, d)obtaining the transgenic plant, wherein the transgenic plant expressesthe coding sequence with below ground tissue preferred expression.
 17. Amethod for manufacturing a synthetic polynucleotide sequence comprisinga sequence identity of at least 97% to SEQ ID NO:1, the methodcomprising: a) isolating a nucleic acid comprising a polynucleotidesequence comprising SEQ ID NO:1; b) producing a plurality ofoligonucleotide primer sequences, wherein the oligonucleotide primersequences bind to the nucleic acid under stringent hybridizationconditions; c) ligating the plurality of oligonucleotide primersequences to synthesize a synthetic polynucleotide sequence; and, d)sequencing the resulting synthetic polynucleotide to confirm that itcomprises at least 97% identity to SEQ ID NO:1.